RS66246B1 - Para-hydroquinone derivatives as vegf, tnf and/or il inhibitors for the treatment of neuroinflammatory diseases - Google Patents

Description

Description

Field of invention

This invention relates generally to novel hydroquinone derivatives, their preparation, and the use of these compounds in methods of treating disorders by administering these compounds to warm-blooded animals in need thereof. The present invention also relates to compounds useful in the prevention and/or treatment of autoimmune, immunological, rheumatological, vascular disorders, ophthalmological disorders, fibrotic disorders, metabolic and gastrointestinal disorders, neuroinflammatory and neurodegenerative diseases, neoplasms and disorders related to cancer, hormonal diseases and immunological disorders resulting from viral and bacterial infectious diseases and their complications.

Basic information about the invention

Some hydroquinone-based derivatives, such as, for example, 2,5-dihydroxybenzenesulfonic acid derivatives and especially calcium dobesylate, ethazilate and persylate are known in the art as active agents for the treatment of male sexual dysfunction and other vascular disorders of endothelial origin, both alone and in combination with other agents. For example, US Patent No. 6,147,112 describes a process for using 2,5-dihydroxybenzenesulfonic acid derivatives, preferably calcium dobesylate, ethamsylate and persylate. Calcium dobesylate or hydroquinone calcium sulfonate, with the chemical name calcium salt of 2,5-dihydroxybenzenesulfonic acid, is sold as Dekium (Delalande) and Doxium (Carrion), and the process for its preparation is described in US patent no.

3,509,207.

There are many drugs containing phenol or catechol groups that suffer from premature metabolism of the hydroxy group during absorption after oral administration. Previous efforts to protect hydroxy groups have generally been unsuccessful because the protecting groups used were either too labile (O=COR or O=CR) or too stable (CH3). Therefore, the objective of the present invention is to provide new hydroquinone derivatives, their pharmaceutically acceptable salts and formulations. These new hydroquinone derivatives have particularly strong anti-fibrotic, anti-inflammatory and anti-angiogenic properties.

International publication VO2018160618A1 discloses certain substituted hydroquinones, 1,4-quinones, catechols, 1,2-quinones, anthraquinones and anthrahydroquinones for use as redox mediators in new technologies, such as in mediator fuel cells or organic mediators.

Richter et al., in Synthesis, 1976, 1976(3), 192-194 disclose bis-N-arylcarbamates and 2,5-dihydroxy-3,6-bis[N-arylcarboxamido]-1,4-benzoquinones and their preparation from 2,5-dihydroxy-1,4-benzoquinone and aryl isocyanates.

Japanese publication JP 51026839A discloses that benzanilides I (R = H, OH) possess antitubercular, analgesic, anti-inflammatory and uric acid excreting activity:

Benzanilides I

US Patent US 3,973,038 also discloses benzoylaniline compounds having the general formula:

where (R1 = OH, acyloxy; R2 = H, OH, lower alkyl, halo, acyloxy; R3 = Cl, Br, lower alkyl; R4 = OH, NH2, lower alkoxy, acyloxy; R5 = H, halo, CO2H, lower alkyl, acyloxy) which has in vivo enzyme inhibition properties, especially histamine deacetylase and xanthine oxidase.

US Patent Application 2008/139632A1 discloses a method for modulating premature translation termination or nonsense-mediated mRNA decay in a cell with an effective amount of hydroxylated 1,2,4-oxadiazole benzoic acid compounds of the formula:

wherein (2 is substituted or unsubstituted aryl, heteroaryl, cycloalkyl, alkyl, alkenyl, heterocycle, arylalkyl or aryloxyalkyl; and X1, X2, X3 and X4 are independently H or OH and at least one of X1, X2, X3 or X4 is OH)

US Patent Application 2013/172333A1 discloses hsp90 inhibitory compounds, which include hydroxylated compounds of 2,4,5-triazole benzoic acid.

International publication WO2010/142766A1 discloses pyrimidine derivatives as zap-70 inhibitors for the treatment or prophylaxis of immune, inflammatory, autoimmune, allergic disorders and immune-mediated diseases.

US Patent Application 2013/121919A1 discloses methods of treating neurodegenerative disorders, conditions associated with accumulated amyloid-beta peptide deposits, Tau protein levels and/or alpha-synuclein accumulations, and cancer in a subject by administering a compound that activates the HAT formula:

wherein (R1 is H, or CF3; R2 is H, Cl or CN; R3 is H, O-methyl, O-ethyl, S-ethyl, O-cyclopentyl, OCH2CH2N(CH3)2 or CH2CH2CH2N(CH3)2; R4 is H;

C(=O)NH-phenyl, wherein the phenyl is substituted with one or more halo or CF3; R5 is H, C1-C5-alkyl, OH, OCH3, O-ethyl, OCH2CH2N(CH3)2, CH2CH2CH2N(CH3)2, SCH2CH2N(CH3)2 or OCH2C(=O)O-alkyl; R6 is H, O-methyl, O-ethyl, OCH2CH2N(CH3)2; and X is CONH, SONH, SO2NH, NHC(=O)NH or NHCO, or a pharmaceutically acceptable salt or hydrate thereof.

Chinese patent application 109687915A1 discloses halogenated benzenediol glucoside compounds of the formula:

where X stands for F, Cl or I.

Korean Patent Application 20060033817 A discloses 3-(2-amino-6-pyridinyl)-4-hydroxyphenyl ketone derivatives of the formula:

wherein A) R 1 is aromatic, heteroaromatic or optionally substituted aromatic or heteroaromatic; B) R 2 is selected from the group consisting of I) cyano; II) a substituent of the formula -C(=O) -X1 -X2, where X1 is a direct bond, oxygen or an optionally substituted amino; X 2 is hydrogen, pyrrolidine, piperidine, piperazine, morpholine, lower alkylamine, hydroxy, optionally substituted lower alkyl, optionally substituted aryl and heteroaryl is selected from the group consisting of; and III) a substituent of the formula -(CH(-X3)) n NX4X5, wherein X3 is hydrogen, halogen and optionally substituted lower alkyl selected from the group consisting of, X4 and X5 are each independently hydrogen, optionally substituted lower alkyl, optionally substituted sulfone and alkyl carbamate, n is an integer from 1 to 5; C) R 3 and R 4 are each independently selected from the group consisting of the following substituents, I) hydrogen; and II) optionally substituted straight chain, branched or cyclic saturated or unsaturated alkyl.

However, no document in the subject field discloses a new class of hydroquinone derivatives of the subject invention, which are particularly useful for the prevention and/or treatment of various diseases including autoimmune, immune, rheumatological, vascular disorders, ophthalmological disorders, fibrotic diseases, metabolic and gastrointestinal disorders, neuroinflammatory and neurodegenerative diseases, cancer-related neoplasms and disorders, hormone-related diseases, and immune disorders resulting from viral and bacterial infectious diseases and their complications.

The invention also relates to the treatment, prevention and reduction of metabolic disorders, such as diabetes and obesity. As blood glucose levels rise after a meal, insulin is secreted and stimulates peripheral tissue cells (skeletal muscle and fat) to actively take up glucose from the blood as a source of energy. Loss of glucose homeostasis as a result of faulty insulin secretion or action typically leads to metabolic disorders such as diabetes, which may be co-triggered or further exacerbated by obesity. Because these conditions are often fatal, strategies are needed to restore adequate clearance of glucose from the bloodstream. Metabolic disorders, especially disorders of glucose and lipid regulation, are becoming more prevalent as the population in industrialized countries ages and sedentary lifestyles become more common. Such disorders are often interrelated and are often predictors or outcomes of each other.

For example, diabetes is caused by a combination of insulin resistance and defective insulin secretion by pancreatic β cells. People with insulin resistance often have abdominal obesity, dyslipidemia, hypertension, glucose intolerance, and a prothrombic state (metabolic syndrome). Consequently, obese individuals overall have a higher risk of developing insulin resistance.

Disruption of the metabolic pathway can therefore cause a myriad of disorders such as hyperlipidemia, obesity, diabetes, insulin resistance, glucose intolerance, hyperglycemia, metabolic syndrome, and hypertension, which in turn can trigger further metabolic dysfunction leading to systemic problems and putting individuals at risk of additional complications and premature morbidity. Glucose and lipid levels are partially regulated by the liver, which plays a role in the synthesis, storage, secretion, transformation, and breakdown of glucose, proteins, and lipids. Disease or traumatic injury can greatly reduce the liver's ability to perform these normal activities. Therefore, most of the clinical manifestations of liver dysfunction originate from cell damage and impairment of normal liver capacities. Liver dysfunction can result from genetic conditions, inflammatory disorders, toxins such as drugs and alcohol, immune disorders, vascular disorders, or metabolic conditions. Regardless of the cause, liver damage can systemically affect the function of metabolic processes and the regulation of blood glucose and serum lipid levels, exacerbating chronic disease states and leading to an increased risk for further disease and morbidity. Both elevated and decreased blood glucose levels trigger hormonal responses designed to restore glucose homeostasis. Low blood glucose levels trigger the release of glucagon from the a-cells of the pancreas. High blood glucose levels cause insulin to be released from the pancreas. ACTH and growth hormones released from the pituitary act to increase blood glucose by inhibiting uptake into extrahepatic tissues.

Glucocorticoids also act to increase blood glucose levels by inhibiting glucose uptake. Cortisol, the major glucocorticoid released from the adrenal cortex, is secreted in response to an increase in circulating ACTH.

The adrenal medulla hormone, epinephrine, stimulates glucose production by activating glycogenolysis in response to stress-stimulo-etabolic disorders that affect glucose and lipid metabolism such as hyperlipidemia, obesity, diabetes, insulin resistance, hyperglycemia, glucose intolerance, metabolic syndrome, and hypertension have long-term health consequences that lead to chronic conditions including cardiovascular disease and premature morbidity. Such metabolic and cardiovascular disorders can be interconnected, exacerbate or trigger each other and create feedback mechanisms that are difficult to interrupt. Current pharmaceutical treatments for metabolic and cardiovascular disorders include combinations of lipid-lowering drugs, hypoglycemic drugs, antihypertensives, diet, and exercise. However, complicated therapeutic regimens can cause problems of polypharmacy due to increased side effects, drug interactions, failure to adhere, and increased medication errors. Therefore, there is a compelling, unmet need in the art to identify novel compounds, formulations, and methods for the safe and effective treatment of metabolic and cardiovascular disorders and conditions associated with metabolic disorders.

Examples of metabolic conditions include, but are not limited to, pain, wound healing, fever, neuroinflammatory and neurodegenerative conditions, inflammation, heat production, homeothermy, triglyceride breakdown, glycolysis, Krebs cycle, fermentation, photosynthesis, metabolic rate, biotic and abiotic stress, secretions, oxidative stress, stress, neoplastic growth, skin conditions, cardiovascular conditions, neuroinflammatory and neurodegenerative conditions, mental and behavioral disorders. Such processes or conditions can occur in a cell, a group of cells or an entire organism.

Recent advances in molecular medicine have led to some improvements in the diagnosis and treatment of neoplastic diseases. Despite this partial success, these pathologies remain a significant challenge. For certain types of cancer, current therapy sometimes fails for several reasons. On the one hand, it is the inherent resistance of tumor cells, their ability to constantly mutate and evade therapy, and on the other hand, the heterogeneity of the tumor environment. It has been shown that tumors of the same type are very different in individual subjects from the point of view of their genomic profile, which indicates the necessity of the so-called "personal" therapies. An even bigger problem is the heterogeneity of mutations in the same tumor, which was recently shown in kidney tumors, and this situation can be expected in other types of tumors as well. For this reason, it is necessary to search for new approaches and an invariable point(s) of intervention that is common to all or most malignant cells in a tumor and that preferably affects the essential functions of cancer cells. It seems that such a point of intervention could be at the metabolic level, for example, mitochondria, ie. organelles that are fundamental for generating energy necessary for all physiological as well as pathophysiological processes in cells. Although tumor cells mainly use so-called aerobic glycolysis for energy production, mitochondrial respiration (ie, oxygen consumption associated with ATP formation) is inherent in most (if not all) types of tumors. Hence, there is an unmet and compelling need in the art to identify novel compounds, formulations, and methods for the safe and effective treatment of such neoplastic disorders.

Summary of the invention

The present invention provides new hydroquinone derivatives, a new preparation process and new key intermediates. The present invention also relates to pharmaceutical compositions containing a therapeutically effective amount of such hydroquinone derivatives and a pharmaceutically acceptable excipient.

The invention further provides the use of these compounds in methods of treatment and/or prevention of angiogenic (vascular), inflammatory and/or fibrotic pathobiology, since they have antifibrotic, antiinflammatory and antiangiogenic effects. The present invention thus ultimately provides the use of these compounds in methods of treatment and/or prevention of diseases or disorders including immunological/rheumatological/vascular disorders, ophthalmic disorders, fibrotic disorders, metabolic and gastrointestinal disorders, neoplasms and cancer-related disorders comprising administering to a subject in need a therapeutically effective dose of the compounds and/or pharmaceutical compositions of the present invention as described above. In particular, the present invention provides a method of treatment and/or prevention of diseases or disorders of autoimmune, immunological, rheumatological, vascular disorders, ophthalmological disorders, fibrotic disorders, metabolic and gastrointestinal disorders, neuroinflammatory and neurodegenerative diseases, neoplasms and disorders related to cancer, hormone-related diseases and immune disorders resulting from viral and bacterial infectious diseases and their complications.

Short description of the pictures

FIGURE 1: (A) Confocal immunofluorescence images of vascular permeability assessed by Evans blue staining in retinal whole mounts in a representative mouse from each experimental group. Arrows indicate the location of extravasation. Scale bar, 30 µm. (B) Quantification of vascular leakage by estimating the number of extravasations per field (0.44 mm2). The effect of Ic-007a eye drops was significant by reducing vascular leakage to the same level as in the non-diabetic control group. *p < 0.01 compared to other groups.

FIGURE 2: (A) Schmidt score is an estimate of compound efficacy based on four histopathological evaluation criteria describing pancreatic injury based on the following scores (number in parentheses) for four parameters. (a) Edema Interstitial edema graded (0) None (1) Interlobular (2) Involved lobules (3) Isolated islands like acinar cells. (b) Inflammatory infiltration = leukocyte infiltration with scores (0) None, (1) < 20%, (2) 20%-50%, (3) > 50%. (c) Parenchymal necrosis = necrosis of acinar cells with scores (0) None, (1) < 5%, (2) 5%-20%, (3) > 20%. (d) Bleeding with scores (0) None, (1) 1-2 points, (2) 3-5 points, (3) > 5 points. The highest Schmidt score = 10 was induced by carulein treatment with the vehicle, and the lowest score = 0 was the unchallenged animals. The Schmidt score is the sum of the a+b+c+d scores and **** means a statistically significant difference with the caerulein-induced group as shown in Figure 2. All products show an improved Schmidt score compared to the caerulein control animals.

FIGURES 3A-D: Figures 3A-B present a list of diseases with major cytokines involved in selected diseases (Akdis M. et al., J Allergi Clin Immunol 2016;

138:984-1010), and Figure 3C presents a portion of the search results with cytokines and genes ranked by results and likely to be involved in the selected disease given as an example of "diabetic retinopathy" after a search at https://www.targetvalidation.org/. In Figure 3D, a search was performed using VEGFA as a cytokine to find a list of diseases where VEGFA is mentioned. Only part of the diseases in which VEGFA is involved is presented in Figure 3D. ref: Open Targets Platform: new developments and updates two years on. Denise Carvalho et al. (Nucleic Acids Research, Volume 47, Issue D1, 8 January 2019, D1056-D1065, https://doi.org/10.1093/nar/gki1133).

FIGURES 4 (A-D): present a table listing the conditions and yields of compounds of type Ia-Id as described in Examples 1.1 to 1.4. Total yield calculated from 2,5-dihydroxyterephthalic acid starting material.

FIGURE 5 (A-C): are a table listing the conditions and yields of compounds of type IIa-IIc as described in Examples 1.5 to 1.7.

FIGURE 6: is a table listing the conditions and yields of compounds of type IIIa and IIIb as described in Examples 1.8 and 1.9.

FIGURE 7: is a table listing the conditions and yields of compounds of type IIIc as described in Example 1.10.

FIGURE 8: is a table listing the deprotection procedures as described in Example 1.12 with respect to Type V compounds.

FIGURE 9: shows fasting blood glucose levels (mean ± stdev of all animals) in the STZ-induced model of diabetes in all animals of each group sampled after 0 days before dosing, and after 7 and 14 days of daily dosing by intravenous or per os routes.

FIGURE 10: shows accelerated wound healing in the STZ-induced model of diabetes in 3 animals per group sampled after 7 days of daily dosing by intravenous or per os routes.

FIGURE 11: Figure 11A shows representative images of lung fibrosis on day 21 of treatment; (A1) uninduced (healthy); A2, Bleomycin induced by vehicle treatment; A3, Bleomycin induced by 100 mg/kg pirfenidone treatment; A4, Bleomycin induced by IVc-059a treatment. Figure 11B shows the Ashcroft score of healthy, untreated bleomycin versus pirfenidone versus IVc-059a.

Detailed description of the invention

Disclosed herein are novel compounds useful for treating subjects suffering from a disease, disorder or dysfunction. Examples of such diseases, disorders or dysfunctions include without limitation autoimmune, metabolic, inflammatory, degenerative and neoplastic disorders, especially inflammatory pathologies, angiogenic diseases and/or fibrotic disorders such as diabetic retinopathy, diabetic nephropathy, ankylosing spondylitis, chronic spondylitis, fibrosis, renal fibrosis, as well as metabolic diseases caused by pancreatic dysfunction.

Hereinafter, unless otherwise indicated, the set of diseases, disorders or dysfunctions that can be treated using the novel compounds disclosed herein are collectively referred to as "medical conditions". The term "subject" as used herein includes any and all organisms and includes the term "patient". A subject treated in accordance with the procedures described herein may be one who has been diagnosed by a physician as suffering from a medical condition. The diagnosis can be made in any convenient way. One skilled in the art will understand that a subject treated in accordance with the present disclosure may have undergone standard tests for the diagnosis of medical conditions. As is known to one of ordinary skill in the art, the clinical features of medical conditions of the type disclosed herein vary according to the pathomechanisms.

Herein, "treatment" refers to the use of the disclosed compounds for therapeutic purposes. Therapeutic treatment may be administered, for example, to a subject suffering from a medical condition to improve or stabilize the subject's condition. Thus, in the claims and embodiments described herein, treatment refers to a subject undergoing, for therapeutic purposes, the methodologies disclosed herein.

Prodrugs as used herein refer to any broad prodrug strategy. For example, prodrug strategies were formally recognized by Adrian Albert in 1958 (Albert, A. Chemical aspects of selective toxicity. Nature, 1958, 182, 421422), but started at the beginning of the last century, as exemplified by methenamine, phenacetin and prontozil. Prodrugs are generally molecules with little or no pharmacological activity but have built-in structural lability, either by accident or design, that allows bioconversion in vivo to the active drug. Conversion can occur by a chemical or enzymatic process or a combination of the two.

Active molecules are often associated with undesirable physicochemical properties that create significant challenges for their delivery to the appropriate biological target. Prodrugs can improve metabolic instability commonly attributed to hepatic metabolism. Similarly, unwanted intestinal drug metabolism can be overcome by selective prodrug strategies. This instability can greatly reduce the total amount of drug that reaches the systemic circulation and its target. Prodrugs can be used to protect active drugs from this first-pass effect by masking a metabolically labile but pharmacologically essential functional group, such as a phenol, to avoid rapid metabolism.

Prodrugs obtained by structural modifications of a drug are designed to affect the inherent physicochemical properties of the molecule to enable its delivery. These developments are not always integrated into the design of new molecules at the discovery stage. Often, analog optimization is the preferred way forward. Applying an early prodrug strategy can result in faster clinical development and, ultimately, drug commercialization.

Many prodrug strategies can be employed to affect the lipophilicity of the parent drug. The lipophilicity of drugs is improved by masking its polar and ionized functionalities with short-chain hydrocarbon substances. Hydrophilic hydroxyl, carboxyl, phosphate or amine and other negatively or positively charged groups are successfully converted into more lipophilic alkyl or aryl esters or N-acyl derivatives, which are rapidly hydrolyzed back to parent drugs in the body by ubiquitous esterases or peptidases.

The present invention therefore provides a compound of formula (1):

whereby:

Ra, Rb = H, C1-4acyl, aryl1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl;

R1 = COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H or CONH-R10;

one of R2 or R3 is H and the other is R5;

R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R5 = R6, R7 or R8;

R6 = CONH-R9, CONHCOR9, CONH(CH2)n-R9 or CONHCH(COOR4)(CH2)kR9, heteroaryl-R9 where k = 0-4;

R7 = benzoheteroaryl substituted with at least one or more groups selected from: COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, OR4, azoles [N-containing 5-membered heterocycles] or fluorine;

R8 = (CH2)mX(CH2)pR9;

X = O, S, SO2, NH, NAc, or N(CH2)qR9;

R9 = aryl or heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, azoles [5-membered heterocycles containing N], fluorine, C=O, NHCO-R10;

R10 = aryl or heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, azoles [N-containing 5-membered heterocycles], fluorine;

wherein aryl R9 and R10 is an aromatic group containing from 6 to 14 carbon atoms, selected from phenyl, naphthyl, biphenyl groups; and heteroaryl R9 and R10 is an aromatic group containing 1 to 14 carbon atoms and one or more nitrogens; and n, m, p and q are independently 1-4.

The subject invention additionally provides a compound of formula (I):

whereby:

Ra, Rb = H, C1-4acyl, aryl1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl;

R1 = COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H or CONH-R10;

one of R2 or R3 is H and the other is R5;

R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R5 = R6, or R7,

R6 = CONH-R9, CONHCOR9, CONH(CH2)n-R9, or CONHCH(COOR4)(CH2)kR9; where k = 0-4;

R7 = benzoheteroaryl substituted with at least one or more groups selected from: COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, OR4, 5-membered heterocycles containing N or fluorine;

R9 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered heterocycles containing N, fluorine, C=O, or NHCO-R10;

R10 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered N-containing heterocycles, or fluorine; wherein aryl R9 and R10 is an aromatic group containing from 6 to 14 carbon atoms, selected from phenyl, naphthyl, biphenyl; and heteroaryl R9 and R10 is an aromatic group containing 1 to 14 carbon atoms and one or more nitrogens; and n is independently 1-4.

The present invention also provides a compound of formula (I):

whereby:

Ra, Rb = H, C1-4acyl, aryl1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl;

R1 = COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H or CONH-R10;

one of R2 or R3 is H and the other is R5;

R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R5 = R8

R8 = (CH2)mX(CH2)pR9;

X = O, S, SO2, NH, NAc, or N(CH2)qR9;

R9 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered heterocycles containing N, fluorine, C=O, or NHCO-R10;

R10 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered heterocycles containing N, fluorine;

wherein aryl R9 and R10 is an aromatic group containing from 6 to 14 carbon atoms, selected from phenyl, naphthyl, biphenyl groups; wherein heteroaryl R9 and R10 is an aromatic group containing 1 to 14 carbon atoms and one or more nitrogens; and n, m, p and q are independently 1-4.

The term "alkyl" refers to a saturated, straight-chain or branched hydrocarbon group containing from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, especially from 1 to 6 (eg 1, 2, 3 or 4) carbon atoms, for example methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl or tert-butyl. Further, the term "alkyl" refers to groups in which one or more hydrogen atoms are replaced by a halogen atom (preferably F or Cl) such as, for example, a 2,2,2-trichloroethyl or trifluoromethyl group.

The term "acyl" refers to the groups (alkyl)-C(O)-, (aryl)-C(O)-, (heteroaryl)-C(O)-, (heteroalkyl)-C(O)- and (heterocycloalkyl)-C(O)-, wherein the group is attached to the parent structure via the carbonyl functionality. In some embodiments, it is a C1-C4 acyl radical that refers to the total number of chain or ring atoms of the alkyl, aryl, heteroaryl, or heterocycloalkyl portion of the acyloxy group plus the carbonyl carbon of the acyl, ie. three other atoms in the ring or chain plus the carbonyl.

The term "aryl" refers to an aromatic group containing one or more rings containing from 6 to 14 ring carbon atoms, preferably from 6 to 10 (especially 6) ring carbon atoms.

The term "aralkyl" or "aryl1-4alkyl" refers to groups containing both aryl and also alkyl, alkenyl, alkynyl and/or cycloalkyl groups as defined above, but are not limited to benzyl, 2-phenylethyl, 3-phenylpropyl and 2-naphth-2-ylethyl. The aralkyl group preferably contains one or two aromatic ring systems (1 or 2 rings) containing from 6 to 10 carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing from 1 or 2 to 6 carbon atoms and/or a cycloalkyl group containing 5 or 6 carbon atoms in the ring.

The term "heteroaryl" refers to an aromatic group containing one or more rings containing from 5 to 14 ring atoms, preferably from 5 to 10 (especially 5 or 6 or 9 or 10) ring atoms, and containing one or more (preferably 1, 2, 3 or 4) oxygen, nitrogen, phosphorus or sulfur atoms in the ring (preferably O, S or N).

Examples are pyridyl (eg 4-pyridyl), imidazolyl (eg 2-imidazolyl), phenylpyrrolyl (eg 3-phenylpyrrolyl), thiazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, oxadiazolyl, thiadiazolyl, indolyl, indazolyl, tetrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolsyloxazolyl, benzylbenzoxazolyl, benzylbenzoxazolyl pyridazinyl, quinolinyl, isoquinolinyl, pyrrolyl, purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3-bifuryl, pyrazolyl (eg 3-pyrazolyl) and isoquinolinyl groups.

The term "benzoheteroaryl" or "benzoheteroaromatic" refers to bicyclic rings containing a phenyl ring fused to a monocyclic heteroaromatic ring. Examples of benzoheteroaryl include benzisoxazolyl, benzoxazolyl, benzisothiazolyl, benzothiazolyl, benzimidazolyl, benzofuryl, benzothienyl (including S-oxide and dioxide), quinolyl, isoquinolyl, indazolyl, indolyl and the like.

The term "azole" refers to a five-membered heteroaryl group having a ring nitrogen atom and between 0 and 3 additional ring heteroatoms selected from N, O or S. Imidazole, oxazole, thiazole and tetrazole are representative azole groups.

The term "cycloalkyl" refers to a saturated or partially unsaturated (for example, a cycloalkenyl group) cyclic group containing one or more rings (preferably 1 or 2) and containing from 3 to 14 ring carbon atoms, preferably from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms. Specific examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl group.

The term "optionally substituted" refers to groups in which at least one, or optionally two, three or more hydrogen atoms may be replaced by fluorine, chlorine, bromine, iodine atoms or by OH, =O, SH, =S, NH2, NOH, N3 or NO2 groups. This term further refers to groups which may be substituted with at least one, two, three or more unsubstituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, heteroalkyl, C3-C18 cycloalkyl, 2-C17 heterocycloalkyl, C4-C20 alkylcycloalkyl, C2-C19 heteroalkylcycloalkyl, C6-C18 aryl, C1-C17 heteroaryl, C7-C20 aralkyl or C2-C19 heteroaryl group. This term further specifically refers to groups which may be substituted with one, two, three or more unsubstituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C2-C9 heterocycloalkyl, C7-C12 alkylcycloalkyl, C2-C11 heteroalkylcycloalkyl, C6-C10 aryl, C1-C9 heteroaryl, C7-C12 aralkyl or C2-C11 heteroaralkyl group.

In accordance with a preferred embodiment, all alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aralkyl and heteroaralkyl groups described herein may be optionally substituted.

Preferably, the present invention provides a hydroquinone derivative of the compound of formula (1), wherein:

(A)

Ra, Rb = H, C1-4acyl, aryl1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl,

R1 = COOR4, R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R2 = H; R3 = R5; and

R5 = R6 = CONH-R9;

or

(B)

Ra, Rb = H, C1-4acyl, aryl1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl,

R1 = COOR4, R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R3 = H; R2 = R5; and

R5 = R6 = CONH-R9;

In a first aspect, the present invention thus includes compounds according to Table 1:

Table 1

Preferably, the subject invention provides a compound of formula (1), wherein:

(A)

Ra, Rb = H;

R1 = SO3H;

R2 = H; R3 = R5; and

R5 = R6 = CONH-R9;

or

(B)

Ra, Rb = H;

R1 = SO3H;

R3 = H; R2 = R5; and

R5 = R6 = CONH-R9;

In another embodiment, the present invention includes compounds according to Table 2:

Table 2

Preferably, the present invention provides a hydroquinone derivative of the compound of formula (1), wherein:

(A)

Ra, Rb = H;

R1 = (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R2 = H; R3 = R5; and

R5 = R6 = CONH-R9;

or

(B)

Ra, Rb = H;

R1 = (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R3 = H; R2 = R5; and

R5 = R6 = CONH-R9;

In a third embodiment, the present invention includes compounds according to Table 3:

Table 3

Preferably, the present invention provides a hydroquinone derivative of the compound of formula (1), wherein:

(A)

Ra, Rb = H, C1-4acyl, aryl1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl;

R1 = CONH-R10; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R2 = H; R3 = R5; and

R5 = R6 = CONH-R9;

or

(B)

Ra, Rb = H, C1-4acyl, aryl1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl;

R1 = CONH-R10; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R3 = H; R2 = R5; and

R5 = R6 = CONH-R9

In a fourth embodiment, the present invention includes compounds according to Table 4:

Table 4

Preferably, the present invention provides a hydroquinone derivative of the compound of formula (I), wherein:

(A)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R2 = H; R3 = R5; and

R5 = R6 = CONHCOR9;

or

(B)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R3 = H; R2 = R5; and

R5 = R6 = CONHCOR9.

In a fifth embodiment, the present invention includes compounds according to Table 5:

Table 5

Preferably, the present invention provides a hydroquinone derivative of the compound of formula (1), wherein

(A)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R2 = H; R3 = R5; and

R5 = R6 = CONH(CH2)n-R9;

or

(B)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R3 = H; R2 = R5; and

R5 = R6 = CONH(CH2)n-R9.

In a sixth embodiment, the present invention includes compounds according to Table 6:

Table 6

Preferably, the present invention provides a hydroquinone derivative of the compound of formula (1), wherein:

Ra, Rb = H;

R1 = SO3H;

R2 = H; R3 = R5; and

R5 = R6 = CONH(CH2)n-R9.

According to a seventh embodiment, the present invention includes compounds according to Table 7 below:

Table 7

Preferably, the present invention relates to a hydroquinone derivative compound of formula (1), wherein:

(A)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R2 = H; R3 = R5; and

R 5 = R 6 = CONHCH(COOR 4 )(CH 2 ) k -R 9 ;

or

(B)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R3 = H; R2 = R5; and

R5 = R6 = CONHCH(COOR4)(CH2)k-R9

According to an eighth embodiment, the present invention includes compounds according to Table 8 below:

Table 8

In a specific embodiment, the present invention provides hydroquinone derivatives of the compound of formula (1), wherein

(A)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; SO3H, (CH2)nSO3H, or CONH-R10; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R2 = H; R3 = R5; and

R5 = R6 = heteroaryl-R9,

or

(B)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; SO3H, (CH2)nSO3H, or CONH-R10;

R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R3 = H; R2 = R5; and

R5 = R6 = heteroaryl-R9

Preferably, the present invention provides a hydroquinone derivative of the compound of formula (1), wherein:

(A)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; SO3H, (CH2)nSO3H, CONH-R10;

R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R2 = H; R3 = R5; and

R5 = R7 = benzoheteroaryl substituted with at least one or more groups selected from: COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, OR4, azoles [N-containing 5-membered heterocycles] or fluorine;

or

(B)

Ra, Rb = H;

R1 = COOR4; (CH2)nCOOR4; SO3H, (CH2)nSO3H, or CONH-R10;

R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R3 = H; R2 = R5; and

R5 = R7 = benzoheteroaryl substituted with at least one or more groups selected from: COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, OR4, azoles [N-containing 5-membered heterocycles] or fluorine;

According to a ninth embodiment, the present invention includes compounds according to Table 9:

Table 9:

Preferably, the present invention provides a hydroquinone derivative of the compound of formula (I), wherein:

(A)

Ra, Rb = H, C1-4acyl, aryl1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl;

R1 = COOR4; (CH2)nCOOR4; SO3H, (CH2)nSO3H, or CONH-R10;

R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R2 = H; R3 = R5; and

R5 = R8 = (CH2)mX(CH2)pR9;

where X = O, S, SO2, NH, NAc or N(CH2)qR9;

or

(B)

Ra, Rb = H, C1-4acyl, aryl1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl;

R1 = COOR4; (CH2)nCOOR4; SO3H, (CH2)nSO3H, or CONH-R10;

R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester);

R3 = H; R2 = R5; and

R5 = R8 = (CH2)mX(CH2)pR9;

where X = O, S, SO2, NH, NAc or N(CH2)qR9.

According to a tenth embodiment, the present invention includes compounds according to Table 10:

Table 10.

The present invention also relates to a process for obtaining a hydroquinone derivative compound of formula (I), which contains:

Step (a): Coupling of a carboxylic acid derived from a hydroquinone, protected at the phenolic functions, with an aniline bearing different functional groups, via the appropriate acyl chloride or using an amide coupling agent such as TCFH in the presence of nmI;

Step (b): Deprotection of the ester usually works by a saponification reaction; and

Step (c): Deprotection of phenolic functions typically by catalytic hydrogenation.

or, alternatively,

Step (d): Reduction of a carboxylic acid derived from a hydroquinone, protected by phenolic functions, to give benzyl alcohol

(e) Activation of the benzylic alcohol function and substitution with a benzylic nucleophile (alcohol, thiol, primary or secondary amine) bearing different functional groups;

(f) Deprotection of the ester works typically by a saponification reaction; and

(g) Deprotection of phenolic functions typically by catalytic hydrogenation.

The process is schematically presented below:

there

The hydroquinone derivative compounds of the present invention can be present as optically active forms (stereoisomers), E/Z isomers, enantiomers, racemates, their diastereoisomers and their hydrates and solvates. Solvate compounds are formed due to the mutual attraction between the molecules of the compound and the inert solvent used. Solvates, e.g. monohydrates, dihydrates or alcoholates.

The hydroquinone derivatives of the present invention may also be present as pharmaceutically acceptable salts, wherein the basic hydroquinone derivatives are modified by preparing their acid or base salts.

Examples of pharmaceutically acceptable salts include, inter alia, salts of mineral or organic acids of basic residues, such as amines; and alkaline or organic salts of acidic residues, such as carboxylic acids and the like.

Pharmaceutically acceptable salts may include conventional non-toxic salts or quaternary ammonium salts suitable for all routes of administration of the compound, for example, from non-toxic organic or inorganic acids. For example, said conventional non-toxic salts include those derived from inorganic acids, such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxylic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-benzoacetoxa, fumaric, toluene sulfonic, methane sulfonic, ethanedisulfonic, oxalic, isethionic, trifluoroacetic acid and the like.

Pharmaceutically acceptable salts are described for cations and anions "Pharmaceutical salts: A summary on doses of salt formers from the Orange Book. Saal C, European Journal of Pharmaceutical Sciences, 2013, 49, 614-623. As examples of cations, we can mention aluminum, arginine, benzathine, calcium, chloroprocaine, choline, diethanolamine, diethylamine, ethanolamine, ethylenediamine, lysine, magnesium, histidine, lithium, meglumine, potassium, procasine, trichysine, potassium, as examples of anions, we can mention acetate, aspartate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, camsylate, carbonate, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glucolate hexanoate, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsilate, nitrate, octanoate, oleate, pamoate, pantothenate, phosphate, polygalacturonate, propionate, stephatesulfate, salinate tartrate, theolate, tosylate. Alternatively, zwitterions can be used as salts such as the free amino acids arginine or lysine as cationic counterions and glutamate or aspartate as anionic counterions. Short peptides (2 to 20 amino acids) as repeating units of arginine or lysine and their combinations with other neutral amino acids. These cationic cell-penetrating peptides (CPPs) can be used as drug penetration enhancers.

Pharmaceutically acceptable salts of the compounds useful in the present invention can, for example, be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, the mentioned salts can be obtained by reacting the free acid or base forms of these compounds with a stoichiometric amount of the corresponding base or acid in water or in an organic solvent, or in a mixture of the two; Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Lists of suitable salts are provided in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p.1418.

Suitable dosages according to the invention and as described herein in various embodiments may vary depending on the condition, age and type of subject, and can be readily determined by those skilled in the art. Total daily doses used in both veterinary and human medicine will conveniently be in the range of 0.01-2000 mg/kg body weight, preferably 0.1-1000 mg/kg body weight, preferably 1-100 mg/kg and these may be administered as single or divided doses, and in addition, the upper limit may also be exceeded when it is determined to be indicated. Such dosage may be tailored to individual requirements in each case including the specific compounds administered, the route of administration, the condition being treated, and the patient being treated. However, the compounds can also be administered as depot preparations (implants, slow-release formulations, etc.) weekly, monthly, or at even longer intervals. In such cases, the dose can be much higher than the daily dose and can be adapted to the form of administration, body weight and specific indications. The appropriate dose can be determined by conducting conventional model tests, preferably animal models. In general, in the case of oral or parenteral administration to adults weighing about 70 kg, a daily dose of about 10 mg to about 10,000 mg, preferably about 200 mg to about 1000 mg, should be appropriate, although the upper limit may be exceeded when indicated. It is understood that the above therapeutically effective doses do not necessarily result from a single administration and usually result from the administration of a number of unit doses. These unit doses may in turn comprise parts of a daily or weekly dose, and therefore the therapeutically effective dose is determined during the treatment (contact) period. For example, for oral administration, the daily dose may be about 0.04 to about 1.0 mg/kg body weight, more preferably about 0.04 to about 0.20 mg/kg/day, more preferably about 0.05 to about 0.15 mg/kg/day, and most preferably about 0.1 mg/kg body weight.

In general, the amount of active substance administered can vary over a relatively wide range to achieve, and preferably maintain, the desired plasma concentration. Unit dosage forms of the active ingredient may contain about 0.1 milligrams to about 15 milligrams thereof. A preferred unit dosage form contains about 0.1 to about 1 milligram of agent and can be administered 2 to 5 times daily. However, it should be noted that other alternative routes such as continuous infusion at a rate designed to maintain the plasma concentration described above are also being considered.

The duration of a particular treatment may also vary depending on the severity of the disease, whether the treatment is intended for an acute manifestation or for prophylactic purposes, and the like. A typical application lasts for a period of about 5 to about 14 days, with a usual course of 7 days.

Courses (cycles) of administration may also be repeated at monthly intervals, or parenteral unit doses may be administered at weekly intervals. Oral unit doses may be administered at intervals of one to several days to provide a specific therapeutically effective dose. The appropriate dose of a compound of the invention will depend on the type of disease being treated, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, the patient's medical history, and the response to the compounds and criteria of the responsible physician.

Determining the appropriate dose or route of administration is clearly within the capabilities of the current physician. Animal experiments provide a reliable guide for determining effective doses for human therapy. Interspecies scaling of effective doses can be done according to the principles established by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics", in Tokikokinetics and New Drug Development, eds Yacobi et al., Pergamon Press, New York, 1989, pp.42-96.

The present invention further provides a pharmaceutical composition comprising (i) a therapeutically effective amount of a hydroquinone derivative compound of formula (1) or a pharmaceutically acceptable salt, or stereoisomers thereof, and (ii) a pharmaceutically acceptable excipient or excipients. Pharmaceutical excipients according to the present invention can be selected from the group consisting of conventional excipients, such as, but not limited to, solubilizers, binders, fillers, disintegrants, lubricants, pH modifiers, permeability enhancers, release modifiers, preservatives, coating agents, carriers, taste masking agents and combinations thereof.

The pharmaceutical composition of the compound of the present invention with one or more pharmaceutical components can be formulated with the aforementioned excipients as liquid drugs, solid or semi-solid drugs and/or gaseous drugs.

When present in solid formulations, they may include, without limitation, granules, pellets, tablets, capsules, powders, films, lozenges, crushable tablets, soluble tablets, disintegrating tablets, dispersible tablets, film-coated tablets, and controlled, extended-release, or modified release formats of any of the aforementioned solid formats. Such formulations may also be in the form of immediate release, delayed release or modified release. Further, immediate release compositions may be conventional, dispersible, chewable, orodispersible or fast-dissolving preparations, and modified release compositions which may contain hydrophilic or hydrophobic, or combinations of hydrophilic and hydrophobic, release rate controlling substances to form a matrix or reservoir or combination of matrix and reservoir systems.

The compositions may be prepared using one or more techniques such as direct mixing, dry granulation, wet granulation, homogenization, milling, micronization, extrusion, and spheronization. The compositions may be presented as uncoated, film coated, sugar coated, powder coated, enteric coated, and modified release coated. When present in liquid formulations, they may include without limitation any liquid forms such as solutions, suspensions, nanosuspensions, dispersions, colloids, emulsions, lotions, creams, ointments, tinctures and freeze-dried compositions. When present in gaseous formulations, they may include without limitation, ground powder or mixture, micronized powder or mixture, nanosuspensions, aerosols, sprays, droplets, mists, atomized solutions or suspensions, and/or atomized vapors.

The pharmaceutical compositions according to the present invention may further contain any additives such as vehicle, binding agents, perfumes, flavors, sweeteners, colors, antiseptics, antioxidants, stabilizers and surfactants, if necessary.

Pharmaceutical compositions and compounds according to the present invention can be formulated for administration by enteral routes, such as oral routes; by parenteral routes via injections such as intravenous, subcutaneous, intraocular, intramuscular or intraperitoneal injections; local routes, such as ocular routes, mucosal and transmucosal routes, including sublingual/buccal routes; as well as pulmonary, nasal, intranasal, intrabronchial, intrapulmonary routes. Ocular routes of administration include topical ocular administration, intraocular, intravitreal or retrobulbar administration.

Preferred formulations of the compounds of the present invention include liquid oral formulations, ophthalmic formulations, and intravenous and intraperitoneal formulations.

Liquid oral formulations are solution and suspension formats for oral administration of drugs that are common, convenient, and considered safe. They can be used to deliver relatively large amounts of medication and are often used in the pediatric population and those who have difficulty swallowing tablets or capsules. Administration through the gastrointestinal tract ensures rapid dissolution and absorption of the drug, but tolerability and interaction with other materials in the tract must be taken into account. Active pharmaceutical ingredient (API) properties can be formulated in simple water-based oral solutions, however, when the API is poorly soluble, formulations should be designed around the molecule to improve solubility and bioavailability. The introduction of surfactants, solvents, buffers and oils increases solubility and reduces precipitation and/or degradation upon interaction with gastric fluids. Where suspensions are required, particle size is often reduced and controlled to improve drug solubility, permeation and ultimately drug absorption. As formulation development progresses, preservatives and flavors are introduced to allow for patient compliance and an appropriate shelf life.

Ocular formulations are ophthalmic formulations that are delivered directly into the eye and are often liquids in the form of solutions or suspensions. Application to the eye avoids metabolism in the gastrointestinal tract and can be used for drugs that are poorly absorbed when administered orally. The eye drops are sterile and isotonic with a nominal pH of 4-8 to avoid eye irritation. Excipients including solubilizing agents, gelling agents, polymers, surfactants, permeation enhancers and cyclodextrins are added to the formulation to improve stability, modify viscosity or increase solubility, permeability and bioavailability of poorly soluble drugs.

Intravenous formulations allow rapid absorption after parenteral drug administration of the entire dose reaching the patient's system for an immediate response. This method of administration avoids metabolism in the gastrointestinal tract and can be used for drugs that are poorly absorbed when administered orally. Injections are usually sterile, isotonic water-based solutions and are designed to be painless when administered. For poorly soluble drugs, formulations are modified with organic co-solvents, surfactants and buffers to increase solubility, and nano-suspension formulations are also acceptable. For those molecules that are unstable in solution, formulations can be lyophilized for dilution at the point of use.

The present invention also provides kits, which contain a composition containing a therapeutically effective dose of one or more compounds of the present invention or formulations thereof, as well as a delivery device for administering the composition or formulation and further containing instructions for use in the package.

In order to prepare the subject pharmaceutical compositions, the active ingredient according to the invention can be mixed with a pharmaceutically acceptable carrier, adjuvant and/or excipient, in accordance with conventional pharmaceutical mixing techniques. Pharmaceutically acceptable carriers that can be used in these compositions include any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, and emulsions, such as

oil/water or water/oil emulsion, and various types of wetting agents. The compositions may additionally contain solid pharmaceutical excipients such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dry milk and the like. Liquid and semi-solid excipients can be selected from glycerol, propylene glycol, polyethylene glycol, water, ethanol and various oils, including oils, oils of animal, vegetable or synthetic origin, etc. Liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols. For examples of carriers, stabilizers and adjuvants, see Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).

The compositions may also include stabilizers and preservatives. Examples of pharmaceutically acceptable solvents that can be used in this composition can be found in reference books such as Handbook of Pharmaceutical Excipients (Ninth Edition, Pharmaceutical Press, London and American Pharmacists Association, Washington, 2020), Aulton's Pharmaceutics, The Design and Manufacture of Medicines. (Fifth Edition, Eds.: Kevin Taylor and Michael Aulton, Elsevier, 2017), "Ullmann's Encyclopedia of Industrial Chemistry, 6th Edition" (various editors, 1989-1998, Marcel Dekker) and in "Pharmaceutical Dosage Forms and Drug Delivery Systems" (ANSEL et al., 1994 and 2011, WILLIAMS & WILKINS).

Non-limiting examples of pharmaceutically acceptable solvents that may be used in this composition include, but are not limited to, propylene glycol (also known as 1,2-dihydroxypropane, 2-hydroxypropanol, methyl ethylene glycol, methyl glycol or propane-1,2-diol), ethanol, methanol, propanol, isopropanol, butanol, glycerol, polyethylene glycol (PEG), glycol, Cremophor EL or any form of polyethoxylated castor oil, dipropylene glycol, dimethyl isosorbide, propylene carbonate, N-methylpyrrolidone, tecofurol glycoethyl fat, esters, and their mixtures.

The compounds according to the invention have shown anti-fibrotic, anti-inflammatory and anti-angiogenic effects. Several of the diseases listed here after involved not just one but two or even three targets. Disease categories exhibiting pre-dominant angiogenic (vascular), inflammatory and/or fibrotic pathobiology.

Therefore, the present invention provides the use of these compounds in the treatment and/or prevention of diseases or disorders of autoimmune, immunological, rheumatological, vascular disorders, ophthalmological disorders, fibrotic disorders, metabolic and gastrointestinal disorders, neuroinflammatory and neurodegenerative diseases, neoplasms. and disorders associated with cancer, hormone-related diseases and immunological disorders resulting from viral and bacterial infectious diseases and complications thereof, which comprises administering to a subject in need thereof a therapeutically effective dose of the compounds and/or pharmaceutical compositions of the present invention as described above.

Pharmaceutical compositions according to the present invention are particularly suitable for a human or animal subject.

Compounds of the invention can target diseases based on their individual cytokine profiles as described in BIODATA for several compounds (see Figures 3A-D). The following Open Targets Platform website (URL: https://www.targetvalidation.org/, Denise Carvalho et al., Nucleic Acids Research, Volume 47, Issue D1, 8 January 2019, D1056-D1065, https://doi.org/10.1093/nar/gki1133) provides a complete picture based on current bibliographic information and is searchable by disease or cytokine.

Two typical examples: (a) a disease-specific search for diabetic retinopathy leads to the following cytokines described as highly correlated with the disease (Figure 3C). The three main cytokines are as an example to search for diabetic retinopathy indicate VEGFA score = 1 (vascular endothelial growth factor A), HFE score = 1 (homeostatic iron regulator) and TNF score = 0.86 (tumor necrosis factor) and (b) search for VEGFA: leads to nervous system disease, vascular disease, eye disease, retinopathy and several other diseases shown in Figure 3D.

To illustrate the potency of the compounds on a small group of inflammatory cytokines, the effects of the compounds on human whole blood (Example 3.6) and their IC50 values on inhibition of LPS-induced inflammation were evaluated (Table 11, in µm).

Table 11:

Immunological/rheumatological/vascular disorders may include for example peritonitis, Kawasaki disease (KD), Takayasu arteritis (TA), microscopic polyangiitis (MP), giant cell arteritis (GCA), antiphospholipid syndrome (APS), Behcet's disease (BD), granulomatosis with polyangiitis (GPA) or Wegener's granulomatosis (VG), eosinophilic granulomatosis with polyangiitis, Churg-Strauss syndrome (EGPA) and rosacea (RO).

Rheumatic disorders particularly affect joint tendons, ligaments and bones with pain, loss of motion and inflammation. This includes many types of arthritis such as osteoarthritis (OA), rheumatoid arthritis (RA), lupus, spondyloarthropathies: ankylosing spondylitis (AS) and psoriatic arthritis (PsA), Sjogren's syndrome, gout, scleroderma, infectious arthritis, juvenile idiopathic arthritis, polymyalgia rheumatica.

Peritonitis is an inflammation of the peritoneum usually caused by a bacterial or fungal infection. There are two types of peritonitis. The first type is spontaneous peritonitis, which can develop as a complication of liver disease, such as cirrhosis, or kidney disease. Secondary peritonitis can be a consequence of abdominal perforation or as a complication of other medical conditions. If left untreated, peritonitis can lead to a severe, potentially life-threatening infection.

Kawasaki disease causes inflammation in the walls of medium-sized arteries throughout the body. First of all, it affects children. Inflammation tends to affect the coronary arteries, which supply blood to the heart muscle. Kawasaki disease is sometimes called mucocutaneous lymph node syndrome because it also affects the lymph nodes that swell during infection, the skin, and mucous membranes in the mouth, nose, and throat.

Takayasu arteritis is a rare type of vasculitis, a group of disorders that cause inflammation of blood vessels. In Takayasu's arteritis, inflammation damages the aorta and its main branches. The disease can lead to narrowed or blocked arteries, or to aneurysms. Takayasu's arteritis can also lead to arm or chest pain, high blood pressure, and eventually heart failure or stroke. Medicines are needed to control inflammation in the arteries and prevent complications.

Granulomatosis with polyangiitis is an uncommon disorder that causes inflammation of the blood vessels in the nose, sinuses, throat, lungs, and kidneys. Formerly called Wegener's granulomatosis, this condition belongs to a group of blood vessel disorders called vasculitis. It slows down the blood flow to some organs. Affected tissues can develop areas of inflammation called granulomas, which can affect the functioning of these organs. Without treatment, the condition can be fatal.

Giant cell arteritis is an inflammation of the lining of the arteries. It most often affects the arteries in the head, especially in the temples. For this reason, giant cell arteritis is sometimes called temporal arteritis. Giant cell arteritis often causes headaches, scalp tenderness, jaw pain, and vision problems. If left untreated, it can lead to blindness.

Antiphospholipid syndrome occurs when the immune system mistakenly creates antibodies that increase the likelihood of blood clotting. This can cause dangerous blood clots in the legs, kidneys, lungs and brain. In pregnant women, antiphospholipid syndrome can also lead to miscarriage and stillbirth. There is no cure for antiphospholipid syndrome. Only available medications can reduce the risk of blood clots. Behcet's disease, also called Behcet's syndrome, is a rare disorder that causes inflammation of the blood vessels. The disease can cause a number of signs and symptoms that may seem unrelated at first. These can include mouth ulcers, eye inflammation, rashes and skin lesions, and sores on the genitals. Immediate treatment can help reduce the symptoms of Behçet's disease and prevent serious complications, such as blindness. Churg-Strauss syndrome is a disorder characterized by inflammation of blood vessels. This inflammation can restrict blood flow to organs and tissues, sometimes permanently damaging them. This condition is also known as eosinophilic granulomatosis with polyangiitis (EGPA). Asthma is the most common sign of Churg-Strauss syndrome. The disorder can also cause other problems, such as hay fever, rashes, gastrointestinal bleeding, pain and numbness in the hands and feet. Churg-Strauss syndrome has no cure. Current medications include steroids and other powerful immunosuppressive drugs to help control symptoms.

Rosacea is a common skin condition that causes redness and visible blood vessels on the face. It can also produce small, red, pus-filled bumps. These signs and symptoms may appear for weeks to months and then disappear for a while. Rosacea can be mistaken for acne, other skin problems, or natural redness. There is no cure for rosacea, but treatment can control and reduce signs and symptoms. Osteoarthritis is the most common form of arthritis, affecting millions of people worldwide. It occurs when the protective cartilage wears away over time. Although osteoarthritis can affect any joint, the disorder most commonly affects the joints of the hands, knees, hips, and spine. Osteoarthritis symptoms can usually be treated, although joint damage cannot be reversed.

Lupus is an autoimmune disease. It causes the immune system to produce proteins called autoantibodies that attack its own tissues and organs, including the kidneys. Lupus nephritis is a common complication in people who have systemic lupus erythematosus (also known as lupus). Lupus nephritis occurs when lupus autoantibodies affect kidney structures. This causes inflammation of the kidneys and can lead to blood in the urine, protein in the urine, high blood pressure, impaired kidney function, or even kidney failure.

Scleroderma is a group of rare diseases that involve hardening and tightening of the skin and connective tissue. There are many different types of scleroderma. In some people, scleroderma affects only the skin. But in many people, scleroderma also damages structures outside the skin, such as blood vessels, internal organs, and the digestive tract (systemic scleroderma). There is no cure for scleroderma.

Sjogren's syndrome is a disorder of the immune system that is recognized by two of the most common symptoms: dry eyes and dry mouth. The condition often accompanies other immune system disorders, such as rheumatoid arthritis and lupus. In Sjogren's syndrome, the mucous membranes and moisture-secreting glands of the eyes and mouth are usually the first to be affected - resulting in decreased tears and saliva. Infectious or septic arthritis is a painful infection in a joint that can be caused by germs that travel through the bloodstream from another part of the body. Septic arthritis can also occur when a penetrating injury, such as an animal bite or trauma, delivers germs directly into the joint. People who have artificial joints are also at risk of septic arthritis. The knees are most commonly affected, but septic arthritis can also affect the hips, shoulders, and other joints. Infection can quickly and severely damage the cartilage and bone inside the joint, so prompt treatment is key.

Juvenile idiopathic arthritis, formerly known as juvenile rheumatoid arthritis, is the most common type of arthritis in children under the age of 16. Juvenile idiopathic arthritis can cause persistent joint pain, swelling, and stiffness. Some children may have symptoms for only a few months, while others may have symptoms for many years. Some types of juvenile idiopathic arthritis can cause serious complications, such as growth problems, joint damage, and eye inflammation.

Polymyalgia rheumatica is an inflammatory disorder that causes pain and stiffness in the muscles, especially in the shoulders and hips. Signs and symptoms of polymyalgia rheumatica usually start quickly and worsen in the morning. Most people who develop polymyalgia rheumatica are over the age of 65. This condition is associated with another inflammatory condition called giant cell arteritis.

Ophthalmic disorders may include, but are not limited to, age-related macular degeneration (AMD or ARMD, dry and wet forms), pterygium (PTE), diabetic retinopathy (DR), diabetic macular edema (DME), Stargardt disease (SD), proliferative vitreoretinopathy (PVR), dry eye syndrome (DIS), endophthalmitis, central serous chorioretinopathy (CSC), retinitis pigmentosa (RP), glaucoma and glaucoma-related complications, and uveitis. (UVE).

Wet macular degeneration is a chronic eye disorder that causes blurred vision or a blind spot in the field of vision. It is usually caused by abnormal blood vessels that leak fluid or blood into the macula. The macula is in the part of the retina responsible for central vision. Wet macular degeneration is one of two types of age-related macular degeneration. Wet type always starts as dry. Dry macular degeneration is more common and less severe. It also causes blurred or reduced central vision, due to thinning of the macula. Dry macular degeneration can first develop in one or both eyes and then affect both eyes. Vision loss is typically central, but remains in peripheral vision.

Diabetic retinopathy is a complication of diabetes that affects the eyes. It is caused by damage to the retinal blood vessels. At first, diabetic retinopathy may cause no symptoms or only mild vision problems. Eventually, it can cause blindness. The condition can develop in anyone with type 1 or type 2 diabetes. DME is a serious eye complication that occurs when microaneurysms protrude from the blood vessel walls, leak or leak fluid and blood into the retina, thereby causing macular edema. Stargadt's disease is an eye disease that causes vision loss in children and young adults. It is a hereditary disease and thus is transmitted to children from parents. Stargardt disease is a form of macular degeneration and is often called juvenile macular degeneration.

PVR is a disease that develops as a complication of rhegmatogenous retinal detachment. PVR occurs in approximately 8-10% of patients undergoing primary retinal detachment surgery and prevents successful surgical repair of rhegmatogenous retinal detachment. PVR can be treated with surgery to reattach the detached retina, but the visual outcome of the surgery is very poor.

Dry eye disease is a common condition that occurs when tears are unable to provide adequate lubrication to the eyes. Tears can be inadequate and unstable for a number of reasons. For example, dry eyes can occur if you don't produce enough tears or if you produce poor quality tears. This tear instability leads to inflammation and damage to the surface of the eye.

Endophthalmitis is an inflammation of the inside of the eye that can occur after an eye injection or surgery. Signs typically include: blurred vision or other vision changes, eye pain, eye redness, eye sensitivity to light, or tearing.

Retinitis pigmentosa (RP) is a group of rare, genetic disorders that involve the breakdown and loss of cells in the retina, which is the light-sensitive tissue that lines the back of the eye. Common symptoms include difficulty seeing at night and loss of side (peripheral) vision.

Glaucoma is a group of eye conditions that damage the optic nerve, the health of which is vital for good vision. This damage is often caused by abnormally high eye pressure. Glaucoma is one of the leading causes of blindness in people over the age of 60. It can occur at any age, but is more common in the elderly. Many forms of glaucoma have no warning signs. The effect is thus gradual with no change in vision until the condition is in an advanced stage.

Uveitis is a form of eye inflammation. It affects the middle layer of tissue in the wall of the eye or uvea. Warning signs of uveitis often appear suddenly and worsen quickly. They include red eyes, pain and blurred vision. Possible causes of uveitis are infection, injury, autoimmune or inflammatory disease. Uveitis can be severe, leading to permanent vision loss.

The compounds and pharmaceutical compositions of the present invention can also be used in the treatment and/or prevention of retinal neurodegenerative diseases selected from the group consisting of diabetic retinopathy, age-related macular degeneration, glaucoma and retinitis pigmentosa.

Neurodegenerative diseases of the retina refer to conditions of the retina characterized by progressive loss of neurons. Diabetic retinopathy, age-related macular degeneration, glaucoma and retinitis pigmentosa are considered retinal diseases in which neurodegeneration plays an essential role.

In accordance with this preferred embodiment, the compounds of the present invention can be combined with other active agents to improve the therapeutic efficacy of the condition being treated, and in particular with a dipeptidyl peptidase-4 inhibitor (DPPIV) or a pharmaceutically acceptable salt thereof, for use in the topical treatment of eyes and/or prevention of neurodegenerative retinal disease. The DPPIV inhibitor can be selected from the group consisting of sitagliptin, saxagliptin, vildagliptin, linagliptin, anagliptin, teneligliptin, alogliptin, trelagliptin, gemigliptin, omarigliptin, its pharmaceutically acceptable salts and mixtures thereof.

The compounds of the present invention can also be combined with anti-VEGF treatment to reduce the number of intraocular anti-VEGF antibody injections required by patients. Administration to patients who have neurodegenerative diseases of the retina, such as diabetic retinopathy, age-related macular degeneration, slows the progression of these diseases and reduces the need for anti-VEGF pharmacotherapy in patients.

Fibrotic disorders may include, but are not limited to, cystic fibrosis, retroperitoneal fibrosis, idiopathic pulmonary fibrosis, combined pulmonary fibrosis and emphysema (CPFE), eosinophilic angiocentric fibrosis, intestinal fibrosis, ovarian fibrosis, ovarian fibrosis, systemic nephrotic fibrosis, oral submucous fibrosis (OSMF), and liver fibrosis.

Cystic fibrosis (CF) is an inherited disorder that causes severe damage to the lungs, digestive system, and other organs in the body. It affects the cells that produce mucus, sweat and digestive juices. Instead of acting as lubricants, secretions clog tubes, ducts and passages, especially in the lungs and pancreas.

Pulmonary fibrosis is a lung disease that occurs when lung tissue becomes damaged and scarred. This thickened, stiff tissue makes it difficult for the lungs to work properly. Scarring associated with pulmonary fibrosis can be caused by a variety of factors. Lung damage caused by pulmonary fibrosis cannot be repaired, but medications and therapies can sometimes help relieve symptoms and improve quality of life.

Emphysema is a lung condition that causes shortness of breath. The alveoli are damaged, and over time the inner walls of the alveoli weaken and rupture, creating larger air spaces instead of many small ones. This reduces the surface area of the lungs and, in turn, the amount of oxygen that reaches the bloodstream. Damaged alveoli do not function properly, which leads to the retention of old air and leaves no room for fresh, oxygen-rich air to enter.

Metabolic and gastro-intestinal disorders may include, for example and without any limitation, complications caused by diabetes: diabetic nephropathy (DN), diabetic retinopathy (DR), diabetic cardiomyopathy (DCDM) or diabetic foot ulcer (DFU). Liver diseases can include non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatic hepatosis (NASH), primary biliary cholangitis (PBC), liver fibrosis or cirrhosis (HF), inflammatory bowel disease (IBD): ulcerative colitis, Crohn's disease, intestinal fibrosis. In addition to diabetic nephropathy, other kidney diseases can include polycystic kidney disease (PKD), chronic kidney disease (CKD), nephrogenic systemic fibrosis (NSF), and pancreatitis (acute and chronic).

Diabetic nephropathy is a serious kidney-related complication of type 1 diabetes and type 2 diabetes. It is also called diabetic kidney disease. About 25% of people with diabetes eventually develop kidney disease.

Diabetic nephropathy affects the kidneys' ability to do their normal job of removing waste products and extra fluid from the body. Over many years, the condition slowly damages the kidneys' delicate filtering system and can progress to kidney failure, also called end-stage kidney disease. Kidney failure is a life-threatening condition.

Nonalcoholic fatty liver disease (NAFLD) is an umbrella term for a range of liver conditions that affect people who drink little or no alcohol. As the name implies, the main feature of NAFLD is too much fat stored in the liver cells.

The most common form of chronic liver disease. Some individuals with NAFLD may develop nonalcoholic steatohepatitis (NASH), an aggressive form of fatty liver disease, which is marked by inflammation of the liver and can progress to advanced scarring (cirrhosis) and liver failure. This damage is similar to the damage caused by heavy alcohol use.

Primary biliary cholangitis is a chronic autoimmune disease in which the bile ducts in the liver are slowly destroyed. When the bile ducts are damaged, bile can back up into the liver and sometimes lead to irreversible cirrhosis. A combination of genetic and environmental factors causes the disease.

IBD describes disorders involving chronic inflammation of the digestive tract. Types of IBD include ulcerative colitis, which involves ulcers along the superficial lining of the colon and rectum, and Crohn's disease, which is characterized by inflammation of the lining of the digestive tract, which can often involve the deeper layers of the digestive tract.

Both ulcerative colitis and Crohn's disease are usually characterized by diarrhea, rectal bleeding, abdominal pain, fatigue, weight loss, and sometimes lead to life-threatening complications.

Cancer-related neoplasms and disorders may include, for example and without limitation, pancreatic cancer: mainly fibrous, renal cell carcinoma, radiation-induced fibrosis, primary myelofibrosis, desmoplasia, fibrosarcoma, hepatocellular carcinoma, retinoblastoma, intraocular and melanoma lymphoma (conjunctival, uveal).

The invention also relates to the treatment, prevention and reduction of viral infections by binding to viral proteins at heparan sulfate binding sites. The compounds of the invention interact with the heparan sulfate binding region of growth factors (FGF, VEGF and other growth factors) and growth factor receptors (FGFR, VEGFR and others). Many viruses are known to interact with mammalian cells using their affinity for heparan sulfate moieties attached to cell membranes. Viruses that have heparan sulfate (HS) binding domains on their surface use this HS affinity as one of the main gateways to access and infect cells and bind to cell-bound heparan sulfate. When attached to HS viruses are able to infect cells using different invasive mechanisms specific to each virus. The strong interaction of the compounds according to the invention with the heparan binding domain of proteins and specifically viral proteins prevents viral infection of blood cells, endothelial cells lining blood vessels and organs, as well as surface epithelial cells present in the mouth, nasal mucosa, and lungs and also in the gastrointestinal tract. Several types of viruses with a high affinity for heparan sulfates are known, such as, but not limited to, respiratory syncytial virus, human metapneumovirus, influenza (H1N1 and similar), human rhinovirus (HRV), rhinosyncytial virus (RV), chikungunya virus, coronavirus such as CoV, SARS-CoV, MERS-Cov, COVID-19 (SARS-CoV-2 and their variants). HS is a necessary cofactor for SARS-CoV-2 infection. HS interacts with the receptor-binding domain of the SARSCoV-2 spike glycoprotein, adjacent to ACE2, shifting the spike structure into an open conformation to facilitate ACE2 binding. Viral and bacterial infections can also include septic shock, inflammation of the eye such as endophthalmitis due to surgery or infection.

Examples

Example 1: synthesis of compounds and intermediates

Example 1.1: molecules of type ia: monoamides, 25 examples

Synthesis of precursors

Scheme 1. Synthetic intermediates KI-1 and KI-6

Procedures:

Synthesis of KI-1 and KI-6 (steps [1], [2])

Diethyl2,5-bis(benzyloxy)terephthalate (KI-0) (Step [1]). Potassium carbonate-325 mesh (326.1 g, 2.4 mol) was added portionwise to a stirred solution of diethyl 2,5-dihydroxyterephthalate (200.0 g, 0.79 mol) in DMF (800.0 mL) at ambient temperature over 15 min. Benzyl bromide (280.0 mL, 2.4 mol) was then added to the reaction flask dropwise over 30 min, and the resulting mixture was heated at 100 °C for 2 h, resulting in the formation of a thick cream-colored precipitate. The reaction mixture was cooled to room temperature and treated with a solution of saturated aqueous ammonium chloride (2 L). The resulting suspension was stirred for 30 min and then filtered. The solid material was washed with saturated aqueous ammonium chloride solution (2 x 200 ml). The solid was then suspended in ethanol (400 mL), filtered and dried in a vacuum oven to give the desired product as a white solid (341.0 g, 0.785 mol, 99.8%)

UPLC-MS (acid procedure, 2 min): rt = 1.36 min, no ionization observed, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 7.51-7.44 (m, 6H), 7.44-7.36 (m, 4H), 7.36-7.28 (m, 2H), 5.17 (s, 4H), 4.29 (q, J = 7.1 Hz), 1.26 (t, J = 7.1 Hz) , 6H).

2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoic acid (KI-1) and 2,5-bis(benzyloxy)terephthalic acid (KI-6) (step [2])

A solution of potassium hydroxide (14.2 g, 0.25 mol) in water (200.0 mL) was rapidly added to a solution of diethyl 2,5-bis(benzyloxy)terephthalate (100 g, 0.23 mol) in 1,4-dioxane (1 L), and the resulting mixture was stirred overnight at room temperature. The solvent was removed in vacuo, resulting in the formation of a white suspension. The crude product was suspended in EtOH (500 mL) and filtered, and the collected solid was dried in a vacuum oven to give pure diethyl 2,5-bis(benzyloxy)terephthalate (23.0 g, 0.053 mol, 23%)

UPLC-MS (acid procedure, 2 min): rt = 1.36 min, no ionization observed, peak area 98%

The ethanol filtrate was concentrated to obtain an oil. Ethyl acetate (500 mL) was added to form a precipitate which was filtered and washed with ethyl acetate (200 mL) to give 2,5-bis(benzyloxy)terephthalic acid (KI-6) as a white solid (18.9 g, 0.042 mol, 18%).

UPLC-MS (acid procedure, 2 min): rt = 1.22 min, m/z 377.1 [MH]-, peak area 95%

<1>H nmR (400 MHz, DMSO-d6) δ 7.52-7.45 (m, 4H), 7.41-7.33 (m, 4H), 7.33-7.26 (m, 4H), 5.13 (s, 4H).

The filtrate was washed with saturated aqueous sodium carbonate solution (200 ml), 1 M aqueous hydrochloric acid solution (500 ml) and brine (500 ml), then dried (Na2SO4), filtered and concentrated to dryness to give 2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoic acid (HI-1) as a white solid (53 g, 0.130 mol, 57.0%).

UPLC-MS (acid procedure, 2 min): rt = 1.22 min, m/z 405.3 [MH]-, peak area 96%

<1>H nmR (400 MHz, DMSO-d6) δ 7.52-7.27 (m, 13H), 5.17 (s, 4H), 4.28 (q, J = 7.1 Hz , 2H), 1.26 (t, J = 7.1 Hz , 3H).

A large-scale protocol for the synthesis of ki-1

A 10 L jacketed vessel was charged with diethyl 2,5-bis(benzyloxy)terephthalate (500.00 g, 1.15 mol), 1,4-dioxane (2.5 L), potassium hydroxide (71.00 g, 1.26 mol), and water (500 mL). The reaction mixture was stirred overnight at room temperature. LCMS analysis showed that the reaction went half-way to completion, also showing a by-product (di-acid) and starting material (di-ester) in the ratio (diester:mono-acid:di-acid = 28:58:14).

(diacid: 4.44 rt, monoacid: 5.08 rt, diester: 5.68 rt). At this stage the solvent was removed in vacuo. The crude product was placed in a 2:1 mixture of ethanol:water (3 L), in a 10 L jacketed vessel, stirred overnight and filtered to give the following:

Substance: (diester:mono-acid:diacid = 86:12:2, 110.00 g, colorless solid)

Filtrates: (diester:mono-acid:diacid 3:63:34)

The filtrates were concentrated in vacuo to give an oil. This oil was taken up in ethyl acetate (2.5 L), stirred overnight in a 10 L jacketed vessel and filtered to give a white solid (150.00 g). Substance:

(diester:mono-acid:diacid = trace: 40:60).

The filtrates were collected, washed with saturated aqueous sodium bicarbonate solution (100 ml), 1N hydrochloric acid solution (200 ml) and brine (200 ml), dried over anhydrous sodium sulfate and filtered.

The solvent was removed under reduced pressure to give the desired product, 2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoic acid (HI-1), as a white solid, which was dried in a vacuum oven at 50 °C for 48 h (225.00 g, 49% yield) (yields vary between 49-65%).

Scheme 2. Synthetic intermediates KI-1Bn

contains 16% KI-0Bn

Procedure:

Synthesis of KI-1Bn:

2,5-bis(benzyloxy)-4-(benzyloxycarbonyl)benzoic acid (KI-1Bn). To a solution of 2,5-dihydroxyter-phthalic acid (1.97 g, 10 mmol) in DMF (20 ml) at 0 °C was added sodium hydride (2.0 g, 50 mmol) with stirring, then benzyl bromide (5.95 ml, 50 mmol, 5 eq.) dropwise over 5 min, and the resulting mixture was kept at room temperature. in 2 hours. The mixture was then heated at 60 °C for 18 h. The reaction mixture was cooled to room temperature and carefully treated with MeOH (5 mL). The mixture was concentrated under reduced pressure and the residue was co-evaporated with heptane (3x). The residue was taken up in DCM (30 ml), the organic phase was washed with 1M HCl (25 ml), the separated aqueous layer was extracted with DCM (3x30 ml) and the combined organic phases were dried (MgSO 4 ) and concentrated. The crude solid was recrystallized from EtOH (~30 mL) to give pure KI-0Bn (2.286 g, 41%). A sample of KI-0Bn (282 mg, 0.5 mmol) was added to a mixture of 1,4-dioxane (10 mL) and lithium hydroxide hydrate (22 mg, 0.52 mmol), and the mixture was heated at 80 °C for 3 d. The mixture was cooled to room temperature, taken up in AcOEt (30 ml) and the solution was washed with 1M HCl (2 ml). The organic phase was dried and concentrated, and the residue subjected to column chromatography to give KI-1Bn monoester contaminated with about 16% KI-0Bn diester.

KI-0Bn:<1>H nmR (400 MHz, CDCl 3 ): δ 7.51 (s, 2H), 7.36-7.30 (m, 20H), 5.34 (s, 4H), 5.11 (s, 4H). KI-1Bn:<1>H nmR (250 MHz, CDCl3): δ 7.86 (s, 1H), 7.61 (s, 1H), 7.41-7.30 (m, 20H), 5.36 (s, 2H), 5.26 (s, 2H), 5.16 (s, 2H).

Synthesis of monoamides

Scheme 3. Synthesis of monoamide Ia

General procedure A for amide coupling [3A]

➢ Coupling via acyl chloride, from KI-1 or KI-6

Model reaction (Ia-013a)

Dimethyl 2-(2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoylamino)isophthalate. 2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoic acid (KI-1, 1.1 g, 2.8 mmol) was dissolved in thionyl chloride (6 mL), and the resulting mixture was stirred for 5 min. A few drops of DMF were added and the resulting mixture was stirred at room temperature for 18 h. Excess thionyl chloride was removed in vacuo and the residue co-distilled with toluene (3 x 20 mL) to give the acyl chloride. A solution of the acyl chloride in DCM (10 mL) was added rapidly to a solution of amine (dimethyl 2-aminoisophthalate, 210 mg, 3.1 mmol) and N,N diisopropylethylamine (0.53 mL, 3.045 mmol) in DCM (10 mL) at room temperature and the reaction was stirred for 18 h. The reaction was diluted with DCM (70 ml) and washed with 1 M aqueous hydrochloric acid (100 ml), water (100 ml), dried (Na 2 SO 4 ), filtered and concentrated to give the crude product, which was purified by silica gel chromatography eluting with a gradient of ethyl acetate (0 to 25%) in isohexane to give the desired product as a white solid (1.24 g, 2.075 mmol, 75%). UPLC-MS (acid procedure, 2 min): rt = 1.37 min; m/z = 598.2 [M+H]<+>, area

peak >95%

<1>H nmR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 8.02 (d, J = 7.8 Hz , 2H), 7.69 (s, 1H), 7.59 - 7.23 (m, 12H), 5.49 (s, 2H), 5.19 (s, 2H), 4.28 (q, J = 7.1 Hz , 2H), 3.71 (s, 6H), 1.26 (t, J = 7.1 Hz , 3H).

General Procedure B for Amide Coupling [3B]

➢ Connection using condensation agent, from KI-1 or KI-6

Model reaction (Ia-001a)

Methyl 2-(2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoylamino)benzoate. 1-Methylimidazole (13.0 mL, 163 mmol) was added to a mixture of 2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoic acid (KI-1, 12.95 g, 32 mmol) and methyl anthranilate (5.729 g, 38 mmol, 1.190 eq) in MeCN (150 mL) at ambient temperature. Chloro-N,N,N'N'-tetramethylformamidinium hexafluorophosphate (TCFH) (10.728 g, 0.038 mol) was added portionwise over 30 min, and the resulting mixture was stirred overnight at room temperature. The reaction mixture was treated with 1 M aqueous hydrochloric acid (700 ml). The resulting suspension was stirred for 10 min and then filtered. The solid material was washed with water (2 x 200 ml). The solid was then suspended in ethanol (200 mL), filtered, washed with ether (100 mL) and dried in a vacuum oven to give the desired product as a white solid (14.7 g, 27 mmol, 86%)

UPLC-MS (acid procedure, 2 min): rt = 1.43 min; m/z = 540.2 [M+H]<+>, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 11.87 (s, 1H), 8.65 (d, J = 8.4 Hz , 1H), 7.98 (dd, J = 8.0, 1.7 Hz , 1H), 7.81 - 7.61 (m, 2H), 7.59 - 7.12 (m, 12H), 5.41 (s, 2H), 5.20 (s, 2H), 4.28 (q, J = 7.1 Hz , 2H), 3.74 (s, 3H), 1.27 (t, J = 7.1 Hz , 3H),0

General deprotection procedures [4], [5]

Saponification

Model reaction (Ia-013a)

2-(2,5-Bis(benzyloxy)-4-carboxybenzoylamino)isophthalic acid A solution of lithium hydroxide (42 mg, 1 mmol) in water (4 mL) was added to a solution of dimethyl 2-(2,5-)bis(benzyloxy)-4-(ethoxycarbonyl)benzoylamino)isophthalate (100 mg,

0.167 mmol) in THF (6 mL) and the resulting mixture was stirred at room temperature for 18 h. Additional LiOH (6 eq.) in water (4 mL) was added to the reaction mixture which was stirred an additional 23 h. After completion, water (50 ml) was added to the reaction and the mixture was washed with ethyl acetate (50 ml). The aqueous layer was acidified with 1 M aqueous hydrochloric acid to pH=2 and extracted with ethyl acetate (2x 50 ml). The organic layers were collected, dried (Na 2 SO 4 ), filtered and concentrated to give the title compound as a white solid (82 mg, 0.151 mmol, 91%).

UPLC-MS (acid procedure, 2 min): rt = 1.02 min; m/z = 542.1 [M+H]<+>, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 13.18 (s, 2H), 11.74 (s, 1H), 8.00 (d, J = 7.8 Hz , 2H), 7.71 (s, 1H), 7.58 - 7.43 (m, 5H), 7.40 - 7.24 (m, 7H), 5.47 (s, 2H), 5.14 (s, 2H).

Debenzylation

Model reaction (Ia-013a)

2-(2,5-Dihydroxy-4-carboxybenzoylamino)isophthalic acid (Ia-013a). Pd/C (94 mg, 10%) was added to a solution of 2-(2,5-bis(benzyloxy)-4-carboxybenzoylamino)isophthalic acid (0.94 g, 1.74 mmol) in a 10:30 mixture of EtOH and DCM. The mixture was placed under hydrogen at atmospheric pressure at room temperature and stirred for 18 h. After completion, the mixture was filtered through celite, which was washed with DCM and MeOH. The filtrate and washings were combined and concentrated under reduced pressure to give the desired product Ia-013a as a yellow solid (644 mg, 1.69 mmol, 98%).

UPLC-MS (acid procedure, 4 min): rt = 0.91 min; m/z =362.0 [M+H]<+>, peak area >98%

<1>H nmR (DMSO-d6) δ: 13.11 (s, 2H), 11.70 (s, 1H), 11.13 (s, 1H), 7.97 (d, J = 7.8 Hz , 2H), 7.47 (s, 1H), 7.41 (s, 1H), 7.38 (t, J = 7.7 Hz , 1H)

General procedure from KI-1Bn

➢ Coupling via acyl chloride

Model reactions: synthesis of Ia-032a

Benzyl 4-(2,5-bis(benzyloxy)-4-(benzyloxycarbonyl)benzoylamino)phenylacetate. To a solution of KI-1Bn (233 mg, 84% purity) in DCM (10 mL) was added SOCl 2 (1.8 mL, 60 eq.) and the mixture was heated at 50 °C for 3 h. The solvents were evaporated and the residue co-evaporated with toluene (3x10 mL) to give the crude acyl chloride (237 mg, 99%). This material was dissolved in DCM (3 mL) and diisopropylethylamine (0.08 mL, 1.15 eq) was added followed by a solution of benzyl 4-aminophenylacetate (92 mg, 0.95 eq) in DCM (3 mL). The final reaction volume was 10 ml. The reaction mixture was stirred at room temperature for 18 hours. The mixture was diluted with DCM (15 mL), washed with saturated aqueous ammonium chloride (12 mL). The separated aqueous phase was extracted with DCM (2x2 ml), the organic phases were combined, dried (MgSO 4 ) and concentrated. The crude product was subjected to column chromatography (Pet. Et./DCM 1:1 then gradient to 100% DCM) to give a first fraction of pure KI-0Bn, and a second fraction containing the pure desired product (237 mg, 86%, corrected for KI-1Bn purity).

<1>H nmR (400 MHz, CDCl3): δ 10.10 (s, 1H), 8.08 (s, 1H), 7.65 (s, 1H), 7.53-7.30 (m, 20H), 7.22-7.13 (br AB, 4H), 5.39, 5.22, 5.21, 5.12 (4s, 4x 2H), 3.61 (s, 2H).

4-(4-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid (Ia-032a). To a solution of the previously prepared precursor (225 mg, 0.33 mmol) in DCM (20 mL) was added 5% Pd on charcoal (72 mg), followed by EtOH (20 mL). The flask was flushed with Ar (3x), then filled with hydrogen (atmospheric pressure) and the mixture was stirred for 2 h at room temperature. The catalyst was removed by filtration through a millipore filter, the filtrate was evaporated, and the residue was dried to give product Ia-032a as a light yellow solid (107 mg, 99%).

<1>H nmR (250 MHz, DMSO-d6): δ 12.28 (br, 1H), 10.92 (s, 1H), 10.44 (s, 1H), 7.65 (br d, 2H), 7.41 (s, 1H), 7.38 (s, 1H), 7.25 (br d, 2H), 3.55 (s, 2H).

For examples:

For conditions and yields: See Figures 4A-D

1) 4-(2-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-001a UPLC-MS (acidic procedure, 2 min): RT = 0.95 min, m/z = 316 [M-H]-, peak area > 97%<1>H nmR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 10.85 (s, 1H), 8.65 (d, J = 8.4 Hz , 1H), 8.01 (dd, J = 8.0, 1.7 Hz , 1H), 7.71 - 7.56 (m, 1H), 7.41 (d, J = 6.9 Hz , 2H), 7.22 (t, J = 7.6 Hz , 1H). HRMS:

[M+H]<+>, calcd. for C15H12NO7: 318.06083, found: 318.06080.

Biological data: Ia-001a: FGF-1 IC50 [µM] = 41; FGF-2 IC50 [µM] = 39; VEGF-A1 IC50 [µM] = 7.3;

VEGFR-inhibition of phosphorylation IC50 [µM] =2.25; PMN ROS [inhibition at 0.3 µm [%] = 48;

PMN ROS inhibition IC50 [µM] = 0.355; Inhibition of neutrophil adhesion [%] = 44.3; Whole blood: GM-CSF IC50 [µM] = >100; IFNγ IC50 [µM] = 30.5; IL-1β IC50 [µM] = 34.4; IL-2 IC50 [µM] = 94.3; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 1.24; IL-9 IC50 [µM] = 5.63; IL-10 IC50 [µM] = >100; IL-12p70 IC50 [µM] = 1.5; IL-13 IC50 [µM] = 26.4; IL-17A IC50 [µM] = 0.1; IL-17F IC50 [µM] = 14.7; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = >100; IL-33 IC50 [µM] = 19.3; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.02; TNFβ IC50 [µM] = 13.2

Diethyl ester:

UPLC-MS (acid procedure, 2 min): rt = 1.39 min, m/z = 372.2 [MH]-, peak area > 98%

<1>H nmR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 11.05 (s, 1H), 9.87 (s, 1H), 8.57 (dd, J = 8.5, 1.2 Hz , 1H), 7.99 (dd, J = 7.9, 1.7 Hz , 1H), 7.65 (ddd, J = 8.7, 7.3, 1.7 Hz , 1H), 7.49 (s, 1H), 7.40 (s, 1H), 7.25 (td, J = 7.6, 1.2 Hz , 1H), 4.35 (m, 4H), 1.33 (m, 6H).

HRMS: [M+H]<+>, calcd. for C19H20NO7: 374.12342, found: 374.12354

Biological data: Ia-001a-E2: FGF-1 IC50 [µM] = 9.2; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 123; VEGFR-inhibition of phosphorylation IC50 [µM] =1.25; PMN ROS [inhibition at 0.3 µm [%] = 51.47; PMN ROS inhibition IC50 [µM] = 2.58; Inhibition of neutrophil adhesion [%] = 50.5

2) 4-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-001aTz:

UPLC-MS (acid procedure, 4 min): rt = 1.17 min; m/z =342.0 [M+H]<+>, peak area >96%

<1>H nmR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 10.90 (s, 2H), 8.47 (d, J = 8.4 Hz , 1H), 7.93 (d, J = 7.9 Hz , 1H), 7.61 (t, J = 7.9 Hz , 1H), 7.50 - 7.32 (m, 3H).

HRMS: [M+H]<+>, calcd. for C15H12N5O5: 342.08329, found: 342.08318

Biological data: Ia-001a-Tz: FGF-1 IC50 [µM] = 6.2; FGF-2 IC50 [µM] = 20; VEGF-A1 IC50 [µM] = 17; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.23; PMN ROS [inhibition at 0.3 µm [%] = 54.33; PMN ROS inhibition IC50 [µM] = 1.54; Inhibition of neutrophil adhesion [%] = 69.75; Whole blood: GM-CSF IC50 [µM] = >100; IFNγ IC50 [µM] = >100; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 7.99; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.75; IL-9 IC50 [µM] = 8.63; IL-10 IC50 [µM] = 26.1; IL-12p70 IC50 [µM] = 32.4; IL-13 IC50 [µM] = 32.1; IL-17A IC50 [µM] = 0.11; IL-17F IC50 [µM] = 46.5; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = >100; IL-33 IC50 [µM] = 23.6; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.63; TNF β IC50 [µM] = 53

Ethyl ester:

UPLC-MS (acid procedure, 2 min): rt = 1.08 min, m/z = 370.1 [M+H]<+>, peak area > 97%

<1>H nmR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 9.88 (s, 1H), 8.46 (d, J = 8.4 Hz , 1H), 7.89 (dd, J = 7.8, 1.6 Hz , 1H), 7.62 (ddd, J = 8.6, 7.4, 1.6 Hz , 1H), 7.47 (s, 1H), 7.42 - 7.34 (m, 2H), 4.37 (q, J = 7.1 Hz , 2H), 1.34 (t, J = 7.1 Hz , 3H).

HRMS: [M+H]<+>, calcd. for C17H16N5O5: 370.11460, found: 370.11458

Biological data: Ia-001a-Tz-El: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =3.8; PMN ROS [inhibition at 0.3 µm [%] = 75.97; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 98.88

Ethyl ester diacetate:

UPLC-MS (acid procedure, 4 min): rt = 1.57 min, m/z = 454.1 [M+H]<+>, peak area > 97%

<1>H nmR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.23 (d, J = 8.6 Hz , 1H), 7.96 (dd, J = 7.9, 1.7 Hz , 1H), 7.84 (s, 1H), 7.73 (s, 1H), 7.64 (t, J = 7.5 Hz) , 1H), 7.43 (td, J = 7.6, 1.2 Hz , 1H), 4.32 (q, J = 7.1 Hz , 2H), 2.34 (s, 3H), 2.18 (s, 3H), 1.32 (t, J = 7.1 Hz , 3H).

HRMS: [M+H]<+>, calcd. for C21H20N5O7: 454.13572, found: 454.13542

Biological data: Ia-001a-Tz-E1-A2: FGF-1 IC50 [µM] = 60; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 49.85; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 98.2

3) 2,5-dihydroxy-4-(2-sulfophenylaminocarbonyl)benzoic acid, compound Ia-001c:

UPLC-MS (acid procedure, 2 min): rt = 0.73 min; m/z =352.0 [M-H]-, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 10.99 (s, 1H), 8.34 (d, J = 8.2 Hz , 1H), 7.74 (dd, J = 7.7, 1.7 Hz , 1H), 7.42 - 7.28 (m, 3H), 7.12 (td, J = 7.5, 1.2 Hz, 1H).

HRMS: [M+H]<+>, calcd. for C14H12NO8S: 354.02781, found: 354.02754

Biological data: Ia-001c: FGF-1 IC50 [µM] = 21; FGF-2 IC50 [µM] = 13; VEGF-A1 IC50 [µM] = 100; VEGFR-inhibition of phosphorylation IC50 [µM] =3.31; PMN ROS [inhibition at 0.3 µm [%] = 40.36; PMN ROS inhibition IC50 [µM] = 2.4;

Inhibition of neutrophil adhesion [%] = 31.33; Whole blood: GM-CSF IC50 [µM] = >100; IFNi IC50 [µM] = 0.44; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 12.8; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.95; IL-9 IC50 [µM] = 46.9; IL-10 IC50 [µM] = 1.9; IL-12p70 IC50 [µM] = 0.44; IL-13 IC50 [µM] = 86.1; IL-17A IC50 [µM] = 0.09; IL-17F IC50 [µM] = 89.7; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = >100; IL-33 IC50 [µM] = >100; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.05; TNF β IC50 [µM] = 29.7

4) 4-(3-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-002a:

<1>H-NMR (250 MHz, DMSO-d6) δ ~13 (v br, 1H), 10.82 (br s, 1H), 10.60 (br s, 1H), 8.38 (s, 1H).7.92 (d, 1H), 7.71 (d, 1H), 7.49 (t, 1H), 7.39 (s, 2H)

Biological data: 1a-002a: FGF-1 IC50 [µM] = 20; FGF-2 IC50 [µM] = 59; VEGF-A1 IC50 [µM] = 71; VEGFR-inhibition of phosphorylation IC50 [µM] =5.7; PMN ROS [inhibition at 0.3 µm [%] = 42.1; PMN ROS inhibition IC50 [µM] = 0.312;

Inhibition of neutrophil adhesion [%] = 27.92

5) 4-(3-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-002aTz:

UPLC-MS (acid procedure, 4 min): rt = 1.13 min; m/z =342.0 [M+H]<+>, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 10.81 (s, 1H), 10.67 (s, 1H), 8.54 (s, 1H), 7.91 - 7.84 (m, 1H), 7.79 (dt, J = 7.8, 1.4 Hz , 1H), 7.60 (t, J = 7.9 Hz, 1H), 7.41 (d, J = 2.2 Hz, 2H).

HRMS: [M+H]<+>, calcd. for C15H12N5O5: 342.08329, found: 342.08317

Biological data: Ia-002a-Tz: FGF-1 IC50 [µM] = 10; FGF-2 IC50 [µM] = 53; VEGF-A1 IC50 [µM] = 57; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 66.61; PMN ROS inhibition IC50 [µM] = 1.86;

Inhibition of neutrophil adhesion [%] = 38.5

6) 2,5-dihydroxy-4-(3-sulfophenylaminocarbonyl)benzoic acid, compound Ia-002c:

<1>H-NMR (250 MHz, DMSO-d6) δ 11.04 (s, 1H), 10.53 (s, 1H), 7.96 (s, 1H), 7.69 (d, 1H), 7.43 (s, 2H) and 7.40-7.28 (m, 2H)

Biological data: Ia-002c: FGF-1 IC50 [µM] = 14; FGF-2 IC50 [µM] = 150; VEGF-A1 IC50 [µM] = 150; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 38.9; PMN ROS inhibition IC50 [µM] = 0.405; Inhibition of neutrophil adhesion [%] = 1.87

7) 4-(4-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-003a:

<1>H-NMR (250 MHz, DMSO-d6) δ 12.8 (br, 1H), 10.73 (s, 1H), 10.68 (s, 1H), 7.94 (d of AB, 2H), 7.84 (d of AB, 2H), 7.39 (s, 1H), 7.34 (s, 1H)

HRMS (AX033)

Biological data: Ia-003a: FGF-1 IC50 [µM] = 12; FGF-2 IC50 [µM] = 34; VEGF-A1 IC50 [µM] = 150; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 44.4; PMN ROS inhibition IC50 [µM] = 0.414;

Inhibition of neutrophil adhesion [%] = 39.1

8) 4-(4-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-003aTz:

UPLC-MS (acid procedure, 2 min): rt = 0.79 min; m/z =342.0 [M+H]<+>, peak area >97%

<1>H nmR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 10.70 (s, 1H), 8.04 (d, J = 8.7 Hz , 2H), 7.96 (d, J = 8.5 Hz , 2H), 7.41 (s, 1H), 7.37 (s, 1H).

HRMS: [M+H]<+>, calcd. for C15H12N5O5: 342.08329, found: 342.08326

Biological data: Ia-003a-Tz: FGF-1 IC50 [µM] = 6.7; FGF-2 IC50 [µM] = 45; VEGF-A1 IC50 [µM] = 13; VEGFR-inhibition of phosphorylation IC50 [µM] =6.4; PMN ROS [inhibition at 0.3 µm [%] = 62.21; PMN ROS inhibition IC50 [µM] = 2.49;

Inhibition of neutrophil adhesion [%] = 30.33; Whole blood: GM-CSF IC50 [µM] = >100; IFNi IC50 [µM] = 2.95; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 43; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 1.41; IL-9 IC50 [µM] = 3.62; IL-10 IC50 [µM] = 6.94; IL-12p70 IC50 [µM] = 1.26; IL-13 IC50 [µM] = 89.6; IL-17A IC50 [µM] = 0.07; IL-17F IC50 [µM] = 83.9; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = 1.49; IL-33 IC50 [µM] = 72.2; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.18; TNF β IC50 [µM] = 55.

9) 2,5-dihydroxy-4-(4-sulfophenylaminocarbonyl)benzoic acid, compound Ia-003c:

<1>H-NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 10.51 (s, 1H), 7.66 (d from AB, 2H), 7.58 (d from AB, 2H), 7.40 (s, 1H), 7.39 (S, 1H)

Biological data: Ia-003c: FGF-1 IC50 [µM] = 35; FGF-2 IC50 [µM] = 150; VEGF-A1 IC50 [µM] = 150; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.4; PMN ROS [inhibition at 0.3 µm [%] = ND; PMN ROS inhibition IC50 [µM]-;

Inhibition of neutrophil adhesion [%] = ND

10) 4-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-004a:

UPLC-MS (acid procedure, 6.5 min): rt = 1.40 min; m/z =332.0 [M-H]-, peak area >96%

<1>H nmR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.37 (s, 1H), 8.20 (d, J = 2.7 Hz , 1H), 7.74 (dd, J = 8.9, 2.8 Hz , 1H), 7.39 (s, 1H), 7.34 (s, 1H), 6.93 (d, J = 8.9 Hz, 1H).

HRMS: [M+H]<+>, calcd. for C15H12NO8: 334.05574, found: 334.05572

Biological data: Ia-004a: FGF-1 IC50 [µM] = 32; FGF-2 IC50 [µM] = 15; VEGF-A1 IC50 [µM] = 28; VEGFR-inhibition of phosphorylation IC50 [µM] =7.7; PMN ROS [inhibition at 0.3 µm [%] = 64.61; PMN ROS inhibition IC50 [µM] = 0;

Inhibition of neutrophil adhesion [%] = 28.38

Ethyl methyl ester:

UPLC-MS (acid procedure, 2 min): rt = 1.16 min, m/z = 376.1 [M+H]<+>, peak area > 95%

<1>H nmR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 10.50 (s, 1H), 10.36 (s, 1H), 9.91 (s, 1H), 8.26 (d, J = 2.7 Hz , 1H), 7.76 (dd, J = 8.9, 2.8 Hz , 1H). 7.43 (s, 1H), 7.36 (s, 1H), 7.01 (d, J = 8.9 Hz , 1H), 4.37 (q, J = 7.1 Hz , 2H), 3.91 (s, 3H), 1.34 (t, J = 7.1 Hz , 3H).

HRMS: [M+H]<+>, calcd. for C18H18NO8: 376.10269, found: 376.10259

Biological data: Ia-004a-E2: FGF-1 IC50 [µM] = 35; FGF-2 IC50 [µM] = 36; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =6.1; PMN ROS [inhibition at 0.3 µm [%] = 85.34; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 97.65

11) 4-(2-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-006a:

UPLC-MS (acid procedure, 2 min): rt = 0.81 min; m/z =334.0 [M+H]<+>, peak area >96%

<1>H nmR (400 MHz, DMSO-d6) δ 13.33 (s, 1H), 11.86 (s, 1H), 10.90 (s, 1H), 9.66 (s, 1H), 8.39 (d, J = 9.0 Hz , 1H), 7.40 (d, J = 6.8 Hz , 2H), 7.03 (dd, J = 9.0, 3.0 Hz, 1H).

HRMS: [M+H]<+>, calcd. for C15H12NO8: 334.05574, found: 334.05562

Biological data: Ia-006a: FGF-1 IC50 [µM] = 89; FGF-2 IC50 [µM] = 224; VEGF-A1 IC50 [µM] = 100; VEGFR-inhibition of phosphorylation IC50 [µM] =11.8; PMN ROS [inhibition at 0.3 µm [%] = 60.56; PMN ROS inhibition IC50 [µM] = 1.2; Inhibition of neutrophil adhesion [%] = 25.33

12) 3-(4-carboxy-2,5-dihydroxybenzamido)phthalic acid, compound Ia-011a:

UPLC-MS (acid procedure, 2 min): rt = 0.69 min; m/z = 360.1 [M-H]-, peak area >89%

<1>H nmR (400 MHz, DMSO-d6) δ 13.46 (s, 2H), 11.37 (s, 1H), 10.97 (s, 1H), 8.33 (dd, J = 8.2, 1.3 Hz , 1H), 7.63 (dd, J = 7.7, 1.3 Hz , 1H), 7.57 (t, J = 7.9 Hz , 1H), 7.52 (s, 1H), 7.44 (s, 1H).

HRMS: [M+H]<+>, calcd. for C16H12NO9: 362.05065, found: 362.05036

Biological data: Ia-011a: FGF-1 IC50 [µM] = 35; FGF-2 IC50 [µM] = 110; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 57.71; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 42.32

13) 2-(4-carboxy-2,5-dihydroxybenzamido)terephthalic acid, compound Ia-012a:

UPLC-MS (acid procedure, 4 min): rt = 1.12 min; m/z = 362.1 [M+H]<+>, peak area >96%

<1>H nmR (400 MHz, DMSO-d6) δ 13.41 (brs, 2H), 12.29 (s, 1H), 10.94 (s, 1H), 9.26 (d, J = 1.7 Hz , 1H), 8.09 (d, J = 8.2 Hz , 1H), 7.73 (dd, J = 8.2, 1.7 Hz, 1H), 7.42 (s, 2H).

HRMS: [M+H]<+>, calcd. for C16H12NO9: 362.05065, found: 362.05046

Biological data: Ia-012a: FGF-1 IC50 [µM] = 38; FGF-2 IC50 [µM] = 36; VEGF-A1 IC50 [µM] = 30; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 59.52; PMN ROS inhibition IC50 [µM] = 1.85;

Inhibition of neutrophil adhesion [%] = 28.5

14) 2-(4-carboxy-2,5-dihydroxybenzamido)isophthalic acid, compound Ia-013a:

UPLC-MS (acid procedure, 4 min): rt = 0.91 min; m/z = 362.0 [M+H]<+>, peak area >98%

<1>H nmR (400 MHz, DMSO-d6) δ 13.11 (s, 2H), 11.70 (s, 1H), 11.13 (s, 1H), 7.97 (d, J = 7.8 Hz , 2H), 7.47 (s, 1H), 7.41 (s, 1H), 7.38 (t, J = 7.7 Hz) , 1H)

HRMS: [M+H]<+>, calcd. for C16H12NO9: 362.05065, found: 362.05041

Biological data: Ia-013a: FGF-1 IC50 [µM] = 29; FGF-2 IC50 [µM] = 18; VEGF-A1 IC50 [µM] = 17; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 38.52; PMN ROS inhibition IC50 [µM] = 2.38;

Inhibition of neutrophil adhesion [%] = 27.67

15) 4-(4-carboxy-2,5-dihydroxybenzamido)phthalic acid, compound Ia-014a:

UPLC-MS (acid procedure, 4 min): rt = 0.98 min; m/z = 362.1 [M+H]<+>, peak area >94%

<1>H nmR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 10.70 (s, 1H), 8.14 (s, 1H), 7.94 - 7.84 (m, 2H), 7.38 (s, 1H), 7.30 (s, 1H).

HRMS: [M+H]<+>, calcd. for C16H12NO9: 362.05065, found: 362.05047

Biological data: Ia-014a: FGF-1 IC50 [µM] = 20; FGF-2 IC50 [µM] = 20; VEGF-A1 IC50 [µM] = 88; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 61.3; PMN ROS inhibition IC50 [µM] = 1; Inhibition of neutrophil adhesion [%] = 9.667

16) 5-(4-carboxy-2,5-dihydroxybenzamido)isophthalic acid, compound Ia-015a:

<1>H nmR (250 MHz, DMSO-d6) δ 10.74 (s, 1H), 8.56 (narrow d, 2H), 8.22 (narrow t, 1H), 7.41 (s, 1H) and 7.37 (s, 1H)

Biological data: Ia-015a: FGF-1 IC50 [µM] = 20; FGF-2 IC50 [µM] = 9.3; VEGF-A1 IC50 [µM] = 19; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.15; PMN ROS [inhibition at 0.3 µm [%] = 43.8; PMN ROS inhibition IC50 [µM] = 0.387;

Inhibition of neutrophil adhesion [%] = 17.38; Whole blood: GM-CSF IC50 [µM] = 2.76; IFNγ IC50 [µM] = >100; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 7.08; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.77; IL-9 IC50 [µM] = 1.47; IL-10 IC50 [µM] = 5.27; IL-12p70 IC50 [µM] = 0.12; IL-13 IC50 [µM] = 0.15; IL-17A IC50 [µM] = 0.05; IL-17F IC50 [µM] = 0.15; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = 57.2; IL-33 IC50 [µM] = 0.24; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.17; TNF β IC50 [µM] = 0.3.

17) 3-(4-carboxy-2,5-dihydroxybenzamido)isonicotinic acid, compound Ia-023a:

UPLC-MS (acid procedure, 4 min): rt = 0.63 min; m/z = 319.1 [M+H]<+>, peak area >93%

<1>H nmR (400 MHz, DMSO-d6) δ 12.17 (s, 1H), 11.03 (s, 1H), 9.78 (s, 1H), 8.46 (d, J = 5.0 Hz , 1H), 7.82 (d, J = 5.0 Hz , 1H), 7.45 (s, 1H), 7.43 (s, 1H).

HRMS: [M+H]<+>, calcd. for C14H11N2O7: 319.05608, found: 319.05610

Biological data: Ia-023a: FGF-1 IC50 [µM] =N.D.; FGF-2 IC50 [µM] = 63; VEGF-A1 IC50 [µM] = 143; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 61.95; PMN ROS inhibition IC50 [µM] = 0; Inhibition of neutrophil adhesion [%] = 13.37

Diethyl ester:

UPLC-MS (acid procedure, 4 min): rt = 1.87 min, m/z = 375.1 [M+H]<+>, peak area > 98%

<1>H nmR (400 MHz, DMSO-d6) δ 11.79 (s, 1H), 11.23 (s, 1H), 9.87 (s, 1H), 9.68 (s, 1H), 8.50 (d, J = 5.0 Hz , 1H), 7.81 (dd, J = 5.1, 0.7 Hz , 1H). 7.53 (s, 1H), 7.41 (s, 1H), 4.46 - 4.27 (m, 4H), 1.41 - 1.25 (m, 6H).

HRMS: [M+H]<+>, calcd. for C18H19N2O7: 375.11867, found: 375.11867

Biological data: Ia-023a-E2: FGF-1 IC50 [µM] =N.D.; FGF-2 IC50 [µM] = 76; VEGF-A1 IC50 [µM] = 141; VEGFR-inhibition of phosphorylation IC50 [µM] =6.6; PMN ROS [inhibition at 0.3 µm [%] = 69.47; PMN ROS inhibition IC50 [µM] = 0; Inhibition of neutrophil adhesion [%] = 34

18) 4-(4-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-032a:

<1>H nmR (250 MHz, DMSO-d6): δ 12.28 (br, 1H), 10.92 (s, 1H), 10.44 (s, 1H), 7.65 (br d, 2H), 7.41 (s, 1H), 7.38 (s, 1H), 7.25 (br d, 2H), 3.55 (s, 2H).

Biological data: Ia-032a: FGF-1 IC50 [µM] =N.D.; FGF-2 IC50 [µM] = 91; VEGF-A1 IC50 [µM] =N.D.; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = ND; PMN ROS inhibition IC50 [µM]-;

Inhibition of neutrophil adhesion [%] = ND

19) 4-(3-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-033a:

UPLC-MS (acid procedure, 6.5 min): rt = 1.71 min; m/z = 332.1 [M+H]<+>, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 10.46 (s, 1H), 7.65 (s, 1H), 7.59 (d, J = 8.1 Hz , 1H), 7.40 (s, 1H), 7.38 (s, 1H), 7.03 (d, J = 7.6 Hz) , 1H), 3.57 (s, 2H).

HRMS: [M+H]<+>, calcd. for C16H14NO7: 332.07647, found: 332.07644

Biological data: Ia-033a: FGF-1 IC50 [µM] = 60; FGF-2 IC50 [µM] = 165; VEGF-A1 IC50 [µM] = 281; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 57.73; PMN ROS inhibition IC50 [µM] = 1.32; Inhibition of neutrophil adhesion [%] = 38

20) 4-(2-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-034a:

UPLC-MS (acid procedure, 2 min): rt = 0.82 min; m/z = 332.1 [M+H]<+>, peak area >98%

<1>H nmR (400 MHz, DMSO-d6) δ 7.78 (d, J = 8.1 Hz , 1H), 7.31 (d, J = 3.0 Hz , 2H), 7.28 (d, J = 7.6 Hz , 2H), 7.15 (t, J = 7.4 Hz , 1H), 3.63 (s, 2H).

HRMS: [M+H]<+>, calcd. for C16H14NO7: 332.07648, found: 332.07641 Biological data: Ia-034a: FGF-1 IC50 [µM] = 79; FGF-2 IC50 [µM] = 14; VEGF-A1 IC50 [µM] = 102; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 49.07; PMN ROS inhibition IC50 [µM] = 2.28; Inhibition of neutrophil adhesion [%] = 15

21) 4-(3,4-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-035a:

UPLC-MS (acid procedure, 2 min): rt = 1.02 min; m/z = 320.1 [M+H]<+>, peak area >91%

<1>H nmR (400 MHz, DMSO-d6) δ 11.56 (s, 1H), 9.21 (t, J = 5.9 Hz , 1H), 8.91 (s, 1H), 8.79 (s, 1H), 7.46 (s, 1H), 7.28 (s, 1H), 6.72 (d, J = 2.1 Hz, 1H), 6.67 (d, J = 8.0 Hz , 1H), 6.57 (dd, J = 8.0, 2.1 Hz , 1H), 4.32 (d, J = 5.8 Hz , 2H).

HRMS: [M+H]<+>, calcd. for C15H14NO7: 320.07648, found: 320.07650

Biological data: Ia-035a: FGF-1 IC50 [µM] = 20; FGF-2 IC50 [µM] = 71; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =36; PMN ROS [inhibition at 0.3 µm [%] = 75.13; PMN ROS inhibition IC50 [µM] = 0.41; Inhibition of neutrophil adhesion [%] = 27

22) 4-(2-(3,4-dihydroxyphenyl)ethylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-035a-2:

UPLC-MS (acid procedure, 2 min): rt = 0.77 min; m/z = 332.0 [M-H]-, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 11.50 (s, 1H), 8.85 (t, J = 5.6 Hz , 1H), 8.76 (s, 1H), 8.66 (s, 1H), 7.41 (s, 1H), 7.28 (s, 1H), 6.67 - 6.60 (m, 2H), 6.48 (dd, J = 8.0, 2.1 Hz , 1H), 3.44 (q, J = 6.8 Hz , 2H), 2.67 (dd, J = 8.6, 6.0 Hz , 2H), δ 11.56 (s, 1H), 9.21 (t, J = 5.9 Hz , 1H), 8.91 (s, 1H), 8.79 (s, 1H), 7.46 (s, 1H), 7.28 (s, 1H), 6.72 (d, J = 2.1 Hz , 1H), 6.67 (d, J = 8.0 Hz , 1H), 6.57 (dd, J = 8.0, 2.1 Hz , 1H), 4.32 (d, J = 5.8 Hz , 2H).

HRMS: [M+H]<+>, calcd. for C16H16NO7: 334.09213, found: 334.09242 Biological data: Ia-035a-2: FGF-1 IC50 [µM] = 10; FGF-2 IC50 [µM] = 65; VEGF-A1 IC50 [µM] = 109; VEGFR-inhibition of phosphorylation IC50 [µM] =10; PMN ROS [inhibition at 0.3 µm [%] = 58.81; PMN ROS inhibition IC50 [µM] = 1.1;

Inhibition of neutrophil adhesion [%] = 21.5

23) 4-(1-carboxy-2-(3,4-dihydroxyphenyl)ethylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-035a-3:

UPLC-MS (acid procedure, 2 min): rt = 0.70 min; m/z = 376.0 [M-H]-, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 12.90 (s, 1H), 11.11 (s, 1H), 8.98 (d, J = 7.4 Hz , 1H), 8.73 (d, J = 13.1 Hz , 2H), 7.45 (s, 1H), 7.31 (s, 1H), 6.61 (dd, J = 5.1, 2.9 Hz , 2H), 6.47 (dd, J = 8.1, 2.1 Hz , 1H), 3.01 (dd, J = 13.9, 4.9 Hz , 1H), 2.96 - 2.84 (m, 1H).

HRMS: [M+H]<+>, calcd. for C17H16NO9: 378.08196, found: 378.08195

Biological data: Ia-035a-3: FGF-1 IC50 [µM] = 34; FGF-2 IC50 [µM] = 207; VEGF-A1 IC50 [µM] = 198; VEGFR-inhibition of phosphorylation IC50 [µM] =10; PMN ROS [inhibition at 0.3 µm [%] = 79.62; PMN ROS inhibition IC50 [µM] = 0.66; Inhibition of neutrophil adhesion [%] = 22

24) 4-(carboxy(3,4-dihydroxyphenyl)methylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-036a:

UPLC-MS (acid procedure, 2 min): rt = 0.62 min; m/z = 362.1 [M-H]-, peak area >92%

<1>H nmR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 9.32 (d, J = 6.7 Hz , 1H), 9.05 (s, 1H), 8.97 (s, 1H), 7.40 (s, 1H), 7.34 (s, 1H), 6.81 (d, J = 2.1 Hz, 1H), 6.75 - 6.64 (m, 2H), 5.29 (d, J = 6.6 Hz , 1H).

HRMS: [M+H]<+>, calcd. for C16H14NO9: 364.06630, found: 364.06615

Biological data: Ia-036a: FGF-1 IC50 [µM] = N.D.; FGF-2 IC50 [µM] = 37; VEGF-A1 IC50 [µM] = 123; VEGFR-inhibition of phosphorylation IC50 [µM] =1.6; PMN ROS [inhibition at 0.3 µm [%] = 87.17; PMN ROS inhibition IC50 [µM] = N.D.;

Inhibition of neutrophil adhesion [%] = 30.77

25) 4-(2-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-053a:

UPLC-MS (acid procedure, 4 min): rt = 1.08 min; m/z = 332.0 [M+H]<+>, peak area >97%

<1>H nmR (400 MHz, DMSO-d6) δ 13.09 (s, 1H), 11.19 (s, 1H), 9.34 - 9.27 (m, 1H), 7.91 (dd, J = 7.8, 1.4 Hz , 1H), 7.55 (dd, J = 7.5, 1.5 Hz , 1H). 7.46 (d, J = 7.9 Hz , 2H), 7.39 (td, J = 7.5, 1.4 Hz , 1H), 7.32 (s, 1H), 4.82 (d, J = 5.9 Hz , 2H).

HRMS: [M+H]<+>, calcd. for C16H14NO7: 332.07648, found: 332.07622

Biological data: Ia-053a: FGF-1 IC50 [µM] = 150; FGF-2 IC50 [µM] = 124; VEGF-A1 IC50 [µM] = 210; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 52.92; PMN ROS inhibition IC50 [µM] = 2.5; Inhibition of neutrophil adhesion [%] = 18.67

Scheme 4b. Synthesis of ethyl 4-amino-2,5-dibenzyloxybenzoate (aniline A)

Aniline A

Protocol: synthesis of aniline A

Ethyl 4-amino-2,5-dibenzyloxybenzoate (Aniline A). To a cooled solution (ice bath) of 2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoic acid (KI-1) (2.0 g, 4.9 mmol) and Et3N (1.6 mL, 11.3 mmol) in THF (25 mL) under an inert atmosphere (N2), was slowly added DPPA (1.1 mL, 5.2 mmol). The mixture was slowly warmed to room temperature and stirred at this temperature for 3 h. Water (8 mL) was then added and the reaction mixture was heated to 70 °C for 2 h. The reaction mixture was poured into saturated sodium hydrogen carbonate solution (250 mL) and stirred for 5 min before being extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give a colorless oil. The residue was purified by flash column chromatography (isohexane/EtOAc 1:0 followed by a gradient to 30% EtOAc) to afford the title compound Aniline A (1.0 g, 53%) as an off-white solid.

UPLC-MS (acid procedure, 2 min): rt = 1.26 min; m/z = 378.2 [M+H]<+>, peak area 94%

<1>H nmR (400 MHz, DMSO-d6) δ 7.54 - 7.46 (m, 4H), 7.44 - 7.36 (m, 4H), 7.35 - 7.25 (m, 3H), 6.46 (s, 1H), 5.65 (s, NH2), 5.06 (s, 2H), 5.02 (s, 2H), 4.16 (q, J = 7.1 Hz , 2H), 1.22 (t, J = 7.1 Hz , 3H).

26) 4-(4-carboxy-2,5-dihydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound Ia-056a:

UPLC-MS (acid procedure, 4 min): rt = 0.90 min; m/z = 348.0 [MH]-, peak area 97%

1H nmR (400 MHz, DMSO-d6) δ 11.28 (brs, 1H), 11.26 (s, 1H), 9.97 (s, 1H), 8.09 (s, 1H), 7.48 (s, 1H), 7.42 (s, 1H), 7.26 (s, 1H).

Diethyl ester:

UPLC-MS (acid procedure, 4 min): rt = 1.86 min; m/z = 404.2 [M-H]-, peak area 92%

<1>H nmR (400 MHz, DMSO-d6) δ 11.48 (brs, 1H), 11.32 (brs, 1H), 10.28 (s, 1H), 10.07 (brs, 1H), 9.88 (s, 1H), 8.12 (s, 1H), 7.58 (s, 1H), 7.39 (s, 1H) 1H), 7.28 (s, 1H), 4.46 - 4.27 (m, 4H), 1.46 - 1.12 (m, 6H).

Example 1.2 - molecules of type Ib: Diamides from diamines, 1 example

Scheme 5. Synthesis of diamide type Ib

Synthetic Procedures: See General Procedures, Steps [3], [4], [5] Example:

For conditions and yields: See Figures 4A-D

27) 3,5-bis(2,5-dihydroxy-4-carboxybenzoylamino)benzoic acid, compound Ib-010a:

UPLC-MS (acid procedure, 4 min): rt = 0.98 min; m/z = 511.0 [M-H]-, peak area >93%

<1>H nmR (400 MHz, DMSO-d6) δ 10.81 (s, 2H), 10.65 (s, 2H), 8.38 (d, J = 2.0 Hz , 1H), 8.12 (d, J = 2.0 Hz , 2H), 7.39 (d, J = 2.3 Hz , 4H).

HRMS: [M+H]<+>, calcd. for C23H17N2O12: 513.07760, found: 513.07736

Biological data: Ib-010a: FGF-1 IC50 [µM] = 14; FGF-2 IC50 [µM] = 3.8; VEGF-A1 IC50 [µM] = 15; VEGFR-inhibition of phosphorylation IC50 [µM] =1.6; PMN ROS [inhibition at 0.3 µm [%] = 71.84; PMN ROS inhibition IC50 [µM] = 1.06;

Inhibition of neutrophil adhesion [%] = 1.5; Whole blood: GM-CSF IC50 [µM] = >100; IFNi IC50 [µM] = 2.22; IL-1β IC50 [µM] = 33.3; IL-2 IC50 [µM] = 2.74; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 1.02; IL-9 IC50 [µM] = 11.58; IL-10 IC50 [µM] = >100; IL-12p70 IC50 [µM] = 0.27; IL-13 IC50 [µM] = >100; IL-17A IC50 [µM] = 0.42; IL-17F IC50 [µM] = >100; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = 67.8; IL-33 IC50 [µM] = 31.3; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.31; TNF β IC50 [µM] = 49.3

Triethyl ester:

UPLC-MS (acid procedure, 2 min): rt = 1.29 min, m/z = 597.1 [M+H]<+>, peak area > 96%

<1>H nmR (400 MHz, DMSO-d6) δ 10.80 (s, 2H), 10.68 (s, 2H), 9.95 (s, 2H), 8.40 (t, J = 2.0 Hz , 1H), 8.15 (d, J = 2.0 Hz , 2H), 7.42 (s, 2H), 7.39 (s, 2H), 4.42 - 4.33 (m, 6H), 1.38 - 1.33 (m, 9H). HRMS: [M+H]<+>, calcd. for C29H29N2O12: 597.17150, found: 597.17131

Biological data: Ib-010a-E3: FGF-1 IC50 [µM] = 26; FGF-2 IC50 [µM] = 226; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.09; PMN ROS [inhibition at 0.3 µm [%] = 36.41; PMN ROS inhibition IC50 [µM] = 6.52; Inhibition of neutrophil adhesion [%] = 79.5; Whole blood: GM-CSF IC50 [µM] = >100; IFNi IC50 [µM] = 2.79; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 36.2; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.24; IL-9 IC50 [µM] = 26.8; IL-10 IC50 [µM] = >100; IL-12p70 IC50 [µM] = 0.84; IL-13 IC50 [µM] = 33; IL-17A IC50 [µM] = 0.52; IL-17F IC50 [µM] = 34.2; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = >100; IL-33 IC50 [µM] = 20; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.98; TNF β IC50 [µM] = 38.2.

Triethyl ester tetraacetate:

UPLC-MS (acid procedure, 2 min): rt = 1.19 min, m/z = 782.1 [M+NH4]<+>, peak area 91%

<1>H nmR (400 MHz, DMSO-d6) δ 10.84 (s, 2H), 8.40 (m, 1H), 8.08 (d, J = 2.0 Hz , 2H), 7.80 (s, 2H), 7.63 (s, 2H), 4.45 - 4.22 (m, 6H), 2.32 (s, 6H), 2.23 (s, 6H), 1.38 - 1.27 (m, 9H).

Biological data: Ib-010a-E3-A4: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.54; PMN ROS [inhibition at 0.3 µm [%] = 54.86; PMN ROS inhibition IC50 [µM] = 1.49; Inhibition of neutrophil adhesion [%] = 96

Example 1.3. - molecules of type Ic: Diamides from diacids, 4 examples

Scheme 6. Synthesis of amides of type Ic with R = R'

Procedures: see general procedures from KI-6 [3], [4], [5]

Examples:

For conditions and yields: See Figures 4A-D

28) N1,N4-bis(2-(1H-tetrazol-5-yl)phenyl)-2,5-dihydroxyterephthalamide, compound Ic-001aTz2:

UPLC-MS (acid procedure, 2 min): rt = 0.97 min; m/z = 485.1 [M+H]<+>, peak area >97%

<1>H nmR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 10.98 (s, 1H), 8.49 (dd, J = 8.4, 1.2 Hz , 1H), 7.90 (dd, J = 7.8, 1.6 Hz , 1H), 7.62 (ddd, J = 8.7, 7.4, 1.6 Hz , 1H), 7.58 (s, 1H), 7.37 (td, J = 7.6, 1.2 Hz , 1H).

HRMS: [M+H]<+>, calcd. for C22H17N10O4: 485.14287, found: 485.17274

Biological data: Ic-001aTz2: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 70; VEGF-A1 IC50 [µM] = 45; VEGFR-inhibition of phosphorylation IC50 [µM] =2.2; PMN ROS [inhibition at 0.3 µm [%] = 60.64; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 97.15; Whole blood: GM-CSF IC50 [µM] = >100; IFNi IC50 [µM] = 1.38; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 9.45; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.52; IL-9 IC50 [µM] = 9.13; IL-10 IC50 [µM] = >100; IL-12p70 IC50 [µM] = 29.1; IL-13 IC50 [µM] = 0.12; IL-17A IC50 [µM] = 0.31; IL-17F IC50 [µM] = 0.15; IL-18 IC50 [µM] = 33.9; IL-21 IC50 [µM] = 32; IL-33 IC50 [µM] = 0.23; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.1; TNF β IC50 [µM] = 0.41.

29) 5-(4-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid, compound Ic-007a:

UPLC-MS (acid procedure, 2 min): rt = 0.80 min; m/z = 469.1 [M+H]<+>, peak area >91%

<1>H nmR (400 MHz, DMSO-d6) δ 8.08 (s, 2H), 7.65 (s, 2H), 7.59 (s, 2H), 7.30 - 6.89 (m, 5H), 6.79 (d, J = 8.8 Hz , 2H).

<13>C nmR (100 MHz, DMSO-d6) δ 172.1, 165.2, 158.3, 150.1, 130.2, 129.2 (CH), 122.8, 122.6 (CH), 117.7 (CH), 117.3 (CH), 113.3.

HRMS: [M+H]<+>, calcd. for C22H17N2O10: 469.08777, found: 469.08739

Biological data: Ic-007a: FGF-1 IC50 [µM] = 13; FGF-2 IC50 [µM] = 5.4; VEGF-A1 IC50 [µM] = 3.2; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.09; PMN ROS [inhibition at 0.3 µm [%] = 57.99; PMN ROS inhibition IC50 [µM] = 2.7; Inhibition of neutrophil adhesion [%] = 57.5; Whole blood: GM-CSF IC50 [µM] = >100; IFNi IC50 [µM] = 1.44; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = >100; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.09; IL-9 IC50 [µM] = 27.1; IL-10 IC50 [µM] = 12.5; IL-12p70 IC50 [µM] = 0.49; IL-13 IC50 [µM] = 30.1; IL-17A IC50 [µM] = 0.04; IL-17F IC50 [µM] = 22.9; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = 1.9; IL-33 IC50 [µM] = 18.4; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.27; TNF β IC50 [µM] = 6.52

Dimethyl ester:

UPLC-MS (acid procedure, 2 min): rt = 1.13 min, m/z = 497.0 [M+H]<+>, peak area > 95%

<1>H nmR (400 MHz, DMSO-d6) δ 11.08 (s, 2H), 10.48 (s, 2H), 10.38 (s, 2H), 8.27 (d, J = 2.8 Hz , 2H), 7.89 - 7.74 (m, 2H), 7.56 (s, 2H), 7.03 (d, J = 8.9 Hz, 2H), 3.93 (s, 6H).

HRMS: [M+H]<+>, calcd. for C24H21N2O10: 497.11907, found: 497.11874

Biological data: Ic-007a-E2: FGF-1 IC50 [µM] = 23; FGF-2 IC50 [µM] = 49; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.25; PMN ROS [inhibition at 0.3 µm [%] = 21.33; PMN ROS inhibition IC50 [µM] = 7.9; Inhibition of neutrophil adhesion [%] = 2

Alternative, large scale synthesis of Ic-007a

a) methyl ester of 5-(4-(3-methoxycarbonyl-4-hydroxyphenylaminocarbonyl)-2,5-dibenzyloxybenzamido)-2-hydroxybenzoic acid

A 5 L flask was charged with 2,5-bis(benzyloxy)terephthalic acid (KI-6, 141.00 g, 0.373 mol), benzyltriethylammonium chloride (850 mg, 3.73 mmol, 0.01 eq.), and chloroform (2.5 L). It is then equipped with a nitrogen inlet, a thermometer, a pressure-equalizing pressure drop funnel, an outlet to an empty Dreschel flask, and a sodium hydroxide bubbler. The mixed solution was heated to 55 °C and thionyl chloride (59 mL, 0.802 mol, 2.15 eq.) was added dropwise over a period of 40 min. The solution was held at 55 °C for 6 h and then allowed to cool to room temperature overnight. An aliquot was sonicated in ethanol for 5 min and then analyzed by LCMS analysis, which showed only diethyl ester. The mixture was transferred to two round bottom flasks and the volatiles were removed in vacuo; the acid chloride was azeotroped with toluene (800 ml in each vessel). A 10 L jacketed vessel was charged with methyl 5-amino-2-hydroxybenzoate (137.00 g, 0.819 mol, 2.2 eq.) and dichloromethane (2.5 L, 18 vol.); the solution was cooled to 15 °C. The acid chloride was placed in dichloromethane (2.5 L, 18 vol.) and added to the stirred reaction. The reaction immediately forms large amounts of solids and mixing becomes difficult; also the reaction is exothermic up to 30 °C. However, with prolonged stirring, the mixture becomes a pink/purple fine suspension. The mixture was stirred at 25 °C for 72 h. The suspension was filtered, washed with dichloromethane (2 L, 14 vol.) and the resulting solid dried in a vacuum oven to give 240.00 g of product (94% yield).

b) 5-(4-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxy benzoic acid, Ic-007a

Saponification: A 5 L 3-necked round bottom flask was charged with freshly ground 5-(4-(3-methoxycarbonyl-4-hydroxyphenylaminocarbonyl)-2,5-dibenzyloxybenzamido)-2-hydroxybenzoic acid methyl ester (150.00 g, 0.22 mol) and isopropanol (1.5 L, 10 vol.). The mixture was heated to 50 °C and tetrabutylammonium hydroxide (444 mL, 0.67 mol., 3 eq.) and water (1.1 L, 7 vol.) were added. After 1 h, some solids remained, so another 60 ml of tetrabutylammonium hydroxide solution was added along with water (120 ml), the mixture was stirred at 50 °C for 6 h, and then allowed to cool to room temperature overnight. More tetrabutylammonium hydroxide (80 mL) was added and the mixture was further stirred at 60 °C for 1 h until a solid remained. Hydrogenolysis: The reaction was degassed (3 cycles with N2), then the catalyst (30.00 g, 10% Pd on C) was added as a suspension in water (100 ml). The reaction was further degassed (3 cycles with N 2 , 2 with H 2 ) and placed under H 2 (balloon pressure) at 50 °C overnight. The reaction was back-charged with hydrogen and kept at 50 °C for another 4 h after which LCMS analysis showed complete reaction. The reaction mixture was degassed (3 cycles with N2), filtered through a pad of celite and washed with hot water/isopropanol (1 L, 1:1, 60 °C). The volume is reduced to ~1.2 l in vacuum. The mixture was transferred to a 5 L flanged flask with water (∼3 L). The flask is equipped with an overhead stirrer (large anchor type) and acidified with hydrochloric acid (35%). A heavy precipitate formed during the addition. The mixture was heated to 55 °C for 6 h and then allowed to cool to room temperature over 72 h. The mixture was filtered and the solid was suspended in 1M aqueous HCl (3 L) and heated at 50 °C for 3 h, before being allowed to cool to room temperature overnight. The mixture was filtered, washed with 1M aqueous HCl (2 L), water (1.5 L) and acetone (1 L). The solid was dried in a vacuum oven to give 100.00 g of product still contaminated with ∼5 mol.% tetrabutylammonium salt (NMR). This solid was stirred in 1M aqueous hydrochloric acid (3 L) at 70 °C for 8 h, then allowed to cool to room temperature overnight. The solid was filtered, washed with water and dried in a vacuum oven. The amount of tetrabutylammonium was reduced to ∼0.62 mol.%. (85.90 g, yield 82%) (brown powder).

30) 2-(2,5-dihydroxy-4-(4-hydroxy-2-carboxyphenylaminocarbonyl)benzamido)-5-hydroxybenzoic acid, compound Ic-009a:

UPLC-MS (acid procedure, 2 min): rt = 0.85 min; m/z = 469.0 [M+H]<+>, peak area >98%

<1>H nmR (400 MHz, DMSO-d6) δ 13.31 (s, 2H), 11.93 (s, 2H), 10.85 (s, 2H), 9.64 (s, 2H), 8.41 (d, J = 9.0 Hz , 2H), 7.55 (s, 2H), 7.39 (d, J = 3.0 Hz) , 2H), 7.03 (dd, J = 9.0, 3.0 Hz , 2H).

HRMS: calc. for C22H17N2O10: 469.08777, found: 469.08755

Biological data: Ic-009a: FGF-1 IC50 [µM] = 32; FGF-2 IC50 [µM] = 202; VEGF-A1 IC50 [µM] = 208; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 72.85; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 72.21

Dimethyl ester:

UPLC-MS (acid procedure, 2 min): rt = 1.03 min, m/z = 497.1 [M+H]<+>, peak area > 92%

<1>H nmR (400 MHz, DMSO-d6) δ 11.69 (s, 2H), 11.01 (s, 2H), 9.75 (s, 2H), 8.36 (d, J = 9.0 Hz , 2H), 7.62 (s, 2H), 7.37 (d, J = 3.0 Hz , 2H), 7.07 (dd, J = 9.0, 3.0 Hz, 2H), 3.87 (s, 6H).

HRMS: calc. for C24H21N2O10: 497.11907, found: 497.11913

Biological data: Ic-009a-E2: FGF-1 IC50 [µM] = 23; FGF-2 IC50 [µM] = 30; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.02; PMN ROS [inhibition at 0.3 µm [%] = 24.12; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 41.05

Scheme 7. Synthesis of amide type Ic with R = R'

Synthetic Procedures: See general procedure from KI-1.

Example:

For conditions and yields: See Figures 4A-D

31) 5-(4-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid, compound Ic-001aTz/004a:

UPLC-MS (acid procedure, 4 min): rt = 1.40 min; m/z = 477.2 [M-H]-, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 10.92 (s, 1H), 10.42 (s, 1H), 8.47 (dd, J = 8.4, 1.2 Hz , 1H), 8.25 (d, J = 2.7 Hz , 1H), 7.98 - 7.88 (m, 1H), 7.76 (dd, J = 8.9, 2.8 Hz , 1H), 7.63 - 7.56 (m, 2H), 7.51 (s, 1H), 7.37 (td, J = 7.6, 1.2 Hz , 1H), 6.97 (d, J = 8.9 Hz , 1H).

HRMS: [M+H]<+>, calcd. for C22H17N6O7: 477.11532, found: 477.11475

Biological data: Ic-001a-Tz/004a: FGF-1 IC50 [µM] = 19; FGF-2 IC50 [µM] = 87; VEGF-A1 IC50 [µM] = 25; VEGFR-inhibition of phosphorylation IC50 [µM] =2.6; PMN ROS [inhibition at 0.3 µm [%] = 82.83; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 96.99; Whole blood: GM-CSF IC50 [µM] = 3.4; IFNγ IC50 [µM] = 0.61; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 4.91; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 3.04; IL-9 IC50 [µM] = 8.48; IL-10 IC50 [µM] = 19.5; IL-12p70 IC50 [µM] = 12.2; IL-13 IC50 [µM] = 1.5; IL-17A IC50 [µM] = 0.02; IL-17F IC50 [µM] = 2.4; IL-18 IC50 [µM] = 46.6; IL-21 IC50 [µM] = 0.3; IL-33 IC50 [µM] = 1.67; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.002; TNF β IC50 [µM] = 3.02

Methyl ester:

UPLC-MS (acid procedure, 2 min): rt = 1.06 min, m/z = 491.1 [M+H]<+>, peak area > 89%

<1>H nmR (400 MHz, DMSO-d6) δ 11.55 (s, 1H), 10.90 (s, 1H), 10.45 (s, 1H), 10.37 (s, 1H), 8.47 (dd, J = 8.4, 1.2 Hz , 1H), 8.30 (d, J = 2.7 Hz , 1H). 7.91 (dd, J = 7.8, 1.6 Hz , 1H), 7.76 (dd, J = 8.9, 2.7 Hz , 1H), 7.63 (ddd, J = 8.7, 7.4, 1.6 Hz , 1H), 7.58 (s, 1H), 7.51 (s, 1H), 7.38 (td, J = 7.6, 1.2 Hz , 1H), 7.02 (d, J = 8.8 Hz , 1H), 3.93 (s, 4H).

HRMS: [M+H]<+>, calcd. for C23H19N6O7: 491.13097, found:

491.13071

Biological data: Ic-001a-Tz/004a-E1: FGF-1 IC50 [µM] = 104; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 35; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.1; PMN ROS [inhibition at 0.3 µm [%] = 73.97; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 97.56

Example 1.4. molecules of type Id: Benzimidazole linked, 1 example

Scheme 8. Synthesis of compounds of type Id

Synthetic procedure: see general procedure from KI-1 [3], [4], [5]

Cyclization step: see Figures 4A-D

Example:

32) 2-(4-carboxy-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-4-carboxylic acid, compound Id-030a:

UPLC-MS (acid procedure, 4 min): rt = 0.99 min; m/z = 313.1 [M-H]-, peak area >97%

<1>H nmR (400 MHz, DMSO-d6) δ 8.10 (d, J = 8.2 Hz , 1H), 8.05 (d, J = 7.7 Hz , 1H), 7.80 (s, 1H), 7.68 (t, J = 8.0 Hz , 1H), 7.62 (s, 1H)

Biodata - unstable in DMSO

Ethyl methyl ester:

UPLC-MS (acid procedure, 4 min): rt = 2.09 min, m/z = 357.1 [M+H]<+>, peak area > 89%

<1>H nmR (400 MHz, DMSO-d6+D2O 10%) δ 7.97 (d, J = 8.0 Hz , 1H), 7.90 - 7.83 (m, 2H), 7.44 - 7.36 (m, 2H), 4.35 (q, J = 7.1 Hz , 2H), 3.94 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H).

HRMS: [M+H]<+>, calcd. for C18H17N2O6: 357.10811, found: 357.10827

Biological data: Id-030a-E2: FGF-1 IC50 [µM] = 115; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.33; PMN ROS [inhibition at 0.3 µm [%] = 0; PMN ROS inhibition IC50 [µM] = 0;

Inhibition of neutrophil adhesion [%] = 43

Example 1.5: molecules of type Ila: Monoamides, 16 examples

Precursor synthesis

Scheme 9a. Synthesis of intermediates KI-2 and KI-7

Procedures:

Synthesis of KI-2 and KI-7

2,6-Di(ethoxycarbonyl)cyclohexane-1,4-dione [Step 1][Ref: Rodriguez et al. Synth. Comm.28 (1998) 2259-69]: 1,3-dichloroacetone (12.5 g, 0.1 mol) in THF (500 ml) was added dropwise over a period of 30 min to a suspension of diethyl 1,3-acetonedicarboxylate (18 ml, 0.1 mol) and potassium carbonate-325 mesh (21.5 g, 0.15 mol) in THF. (1 L) at reflux temperature. After 2 h, the reaction was complete and the mixture was cooled to room temperature and then filtered through celiteⓒ; the solid residue was washed with THF (200 mL), the solvent was then removed under reduced pressure. The crude product was purified by flash column chromatography (Hexane/EtOAc 0 to 20%) to afford the title compound (9.3 g, 37% yield).

UPLC-MS (acid procedure, 2 min): rt = 1.01 min; m/z = 257.1 [M+H]-, peak area >74%

<1>H nmR (400 MHz, DMSO-d6) Complex mixture of enol and cis/trans isomer δ 12.10 (s), 4.23 (q, J = 7.1 Hz ), 4.11 (m), 3.85 - 3.77 (m), 3.70 (s), 3.10 -2.89 (m), 2.89 - 2.80 (m). 2.62 - 2.58 (m), 1.25 (t, J = 7.1 Hz), 1.22 - 1.15 (m, 9H).

Diethyl 2,5-dihydroxyisophthalate [Step 2]: [Protocol adapted from Zhong et al.: Chem Eur. J.25 (2019) 8177-8169]: To a stirred solution of 2,6-di(ethoxycarbonyl)-cyclohexane-1,4-dione (5.0 g, 20 mmol) in AcOH (17 ml) at room temperature was added NBS (3.5 g, 20 mmol) in portions over 30 min. After an additional 20 minutes, the reaction was quenched by the addition of water (100 mL). The desired product is isolated as a solid. After filtration and drying, the title compound was isolated as a white solid (4.6 g, 93% yield).

UPLC-MS (acid procedure, 2 min): rt = 1.00 min; m/z = 253.1 [M-H]-, peak area >90%

<1>H nmR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.57 (s, 1H), 7.39 (s, 2H), 4.32 (q, J = 7.1 Hz , 4H), 1.31 (t, J = 7.1 Hz , 6H).

Diethyl 2,5-bis(benzyloxy)isophthalate [Step 3]: To a suspension of diethyl 2,5-dihydroxyisophthalate (19.0 g, 75 mmol) and potassium carbonate-325 mesh (82.6 g, 598 mmol) in DMF (83 mL) was added dropwise benzyl bromide (43.0 mL, 362 mmol) over 10 min. The reaction mixture was heated at 100 °C for 2 h. After cooling to room temperature, the solvent was removed under reduced pressure. Then water (500 ml) was added to the residue. The mixture was extracted with EtOAc (3 x 250 mL), the combined organic layers were washed with brine (300 mL), dried over Na 2 SO 4 , filtered and then concentrated under reduced pressure. The crude material was purified by flash column chromatography (Hexane/EtOAc 0 to 15%) to afford the title compound (29.4 g, 90% yield).

UPLC-MS (acid procedure, 2 min): rt = 1.38 min; m/z = 435.2 [M+H]<+>, peak area >90%

<1>H nmR (400 MHz, DMSO-d6) δ 7.50 (s, 2H), 7.49 - 7.45 (m, 2H), 7.44 -7.37 (m, 5H), 7.37 - 7.29 (m, 3H), 5.17 (s, 2H), 4.95 (s, 2H), 4.26 (q, J = 7.1 Hz , 4H), 1.22 (t, J = 7.1 Hz , 6H).

2,5-Bis(benzyloxy)-3-(ethoxycarbonyl)benzoic acid (KI-2) [Step 4]: A solution of potassium hydroxide (4.4 g, 79 mmol) in water (66 mL) was rapidly added to a solution of diethyl 2,5-bis(benzyloxy)isophthalate (31.3 g, 72 mmol) in 1,4-dioxane (430 mL). The reaction mixture was stirred at room temperature for 1.5 h. 1,4-dioxane was removed under reduced pressure, then saturated aqueous Na2CO3 (1 L) was added, the aqueous layer was extracted with EtOAc (3x 500 ml), dried over Na2SO4, filtered and the solvent was removed in vacuo. The residue was purified by filtration over a pad of silica using hexane/EtOAc (1/1), then EtOAc (1% vol./vol. AcOH) as eluent to give two distinct fractions:

Starting material: Diethyl 2,5-bis(benzyloxy)isophthalate (20.4 g, 65% yield).

UPLC-MS (acid procedure, 2 min): rt = 1.38 min; m/z = 435.2 [M+H]<+>, peak area >78%

<1>H nmR (400 MHz, DMSO-d6) δ 7.50 (s, 2H), 7.49 - 7.45 (m, 2H), 7.44 -7.37 (m, 5H), 7.37 - 7.29 (m, 3H), 5.17 (s, 2H), 4.95 (s, 2H), 4.26 (q, J = 7.1 Hz , 4H), 1.22 (t, J = 7.1 Hz , 6H).

Desired product, 2,5-bis(benzyloxy)-3-(ethoxycarbonyl)benzoic acid as a white fluffy solid, (KI-2) (3.5 g, 12% yield).

UPLC-MS (acid procedure, 2 min): rt = 1.22 min; m/z = 405.1 [M-H]-, peak area >90%

<1>H nmR (400 MHz, DMSO-d6) δ 13.30 (s, 1H), 7.48 (t, J = 3.0 Hz , 2H), 7.46 - 7.42 (m, 5H), 7.39 (m, 3H), 7.37 - 7.31 (m, 2H), 5.17 (s, 2H), 4.96 (s, 2H), 4.25 (q, J = 7.1 Hz , 2H), 1.22 (t, J = 7.1 Hz , 3H).

The original saturated aqueous solution of Na2CO3 was then acidified to pH~7 using concentrated hydrochloric acid. Then extracted with EtOAc (2 × 500 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a mixture of KI-2 and KI-7 (3.0 g, 11% yield).

UPLC-MS (acid procedure, 2 min): rt = 1.00 min; m/z = 377.1 [M-H]-, peak area 25%, rt = 1.22 min; m/z = 405.1 [M-H]-, peak area 56%

<1>H nmR (400 MHz, DMSO-d6) δ 13.23 (s, 2H), 7.52 - 7.29 (m, 12H), 5.17 (s, 2H), 4.97 (s, 2H).

Scheme 9b. Alternative synthesis of KI-7

Formic acid

[Step 3]

Pinnick oxidation

[Step 5]

2,6-Dimethyl-1,4-phenylene diacetate [Step 1]: To a stirred solution of 2,6-dimethylhydroquinone (5.0 g, 36 mmol) in pyridine (10 mL) was rapidly added acetic anhydride (10 mL). The solution was stirred at room temperature for 18 h. The solvent was then removed under reduced pressure and the residue was dissolved with EtOAc (250 mL) and then washed with aqueous hydrochloric acid (1 M, 250 mL), saturated aqueous NaHCO 3 (250 mL) and water (250 mL), dried over Na 2 SO 4 , filtered and the solvent removed in vacuo to give the title compound (7.4 g, 92%) as a white solid. solid substances.

UPLC-MS (acid procedure, 2 min): rt = 1.07 min; m/z = 240.2 [M+NH4]<+>, peak area >90%

<1>H nmR (400 MHz, DMSO-d6) δ 6.88 (HAr, 2H), 2.37 - 2.30 (m, 3H), 2.27 - 2.21 (m, 3H), 2.12 - 2.02 (m, 6H).

2,6-bis(dibromomethyl)-1,4-phenylene diacetate [Step 2]: To a stirred solution of 2,6-dimethyl-1,4-phenylene diacetate (6.34 g, 29 mmol) in 1,2-dichloroethane (130 mL) was added sequentially 2,2'-Azobis(2-methylpropionitrile) (AIBN) (0.93 g, 29 mmol).

6 mmol) and N-bromosuccinimide (NBS) (25.39 g, 143 mmol). The solution was stirred under reflux for 24 h. 2,2'-Azobis(2-methylpropionitrile) (AIBN) (0.93 g, 6 mmol) and N-bromosuccinimide (NBS) (5.08 g, 28.6 mmol) were added and the reaction mixture was stirred under reflux for an additional 24 h. The reaction was cooled to room temperature and the residual solid was removed by filtration and washed with DCM (250 mL). The filtrate was washed with saturated aqueous sodium hydrogen carbonate (250 ml), aqueous hydrochloric acid (1 M, 250 ml) and brine (250 ml).

The organic layer was then dried with sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (9.38 g, 61%) as an orange oil.

UPLC-MS (acid procedure, 2 min): rt = 1.23 min; m/z = 553.1 [M+NH4]<+>, peak area >88%

<1>H nmR (400 MHz, DMSO-d6) δ 7.69 (s, 2H), 7.31 (s, 2H), 2.49 (s, 3H), 2.32 (s, 3H).

2,5-Dihydroxyisophthalaldehyde [Step 3]: To a stirred suspension of 2,6-bis(dibromomethyl)-1,4-phenylene diacetate (3.89 g, 7.23 mmol) in formic acid (50 mL) was added water (5 mL). The mixture was stirred under reflux for 18 h. The reaction mixture was then slowly poured into a saturated aqueous solution of sodium hydrogen carbonate (300 ml). The precipitate formed was then isolated by filtration to give the title compound (0.94 g, 78%) as a brown solid.

UPLC-MS (acid procedure, 2 min): rt = 0.74 min; m/z = 165.0 [M-H]-, peak area >98%

2,5-Bis(benzyloxy)isophthalaldehyde [Step 4]: Potassium carbonate-325 mesh (2.34 g, 17.0 mmol) was added to a stirred solution of 2,5-dihydroxyisophthalaldehyde (0.94 g, 5.7 mmol) in DMF (6 mL) at ambient temperature. Benzyl bromide (2.0 mL, 17.0 mmol) was then added to the reaction flask, and the resulting mixture was heated at 100 °C for 18 h. The reaction mixture was cooled to room temperature and treated with saturated aqueous ammonium chloride solution (100 ml). The resulting suspension was stirred for 30 min and then filtered. The solid material was washed with saturated aqueous ammonium chloride solution (2 x 50 ml). The solid was then triturated with ethanol (5 mL), dried under suction to give the desired product as a brown solid (1.58 g, 68%)

UPLC-MS (acid procedure, 2 min): rt = 1.28 min, no ionization observed, peak area 78%

<1>H nmR (400 MHz, DMSO-d6) δ 10.14 (s, 2H), 7.63 (s, 2H), 7.53 - 7.28 (m, 10H), 5.23 (s, 2H), 5.21 (s, 2H).

2,5-Bis(benzyloxy)isophthalic acid (KI-7) [Step 5]: 2,5-bis(benzyloxy)isophthalaldehyde (1.58 g, 4.5 mmol) was dissolved in a solution of 2-methyl-2-butene in THF (2.0 M, 25 mL). A solution of sodium chlorite (5.1 g, 45.0 mmol) and potassium dihydrogen phosphate (4.6 g, 33.8 mmol) in water (25 mL) was then added and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was then poured into saturated aqueous NaHCO 3 (400 mL). The aqueous layer was washed with EtOAc (3 × 100 mL) followed by acidification with conc. hydrochloric acid (pH-1). The aqueous layer was then extracted with DCM (2 x 150 ml) and Et2O (2 x 200 ml), the combined organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was then triturated with n-pentane (3 × 50 mL) to give the title compound KI-7 as a white solid (0.98 g, 58%).

UPLC-MS (acid procedure, 2 min): rt = 1.03 min, m/z = 379.1 [M+H]<+>, peak area >96%

<1>H nmR (400 MHz, DMSO-d6) δ 13.23 (s, 2H), 7.57 - 7.28 (m, 12H), 5.17 (s, 2H), 4.97 (s, 2H).

Scheme 9c. Synthesis of KI-2Ac2

Methyl 2,5-diacetoxy-3-methylbenzoate.3-methylsalicylic acid was oxidized with persulfate as described by Nudenberg et al. (J. Org. Chem. 1943, 8, 500-508). 3-Methylsalicylic acid (15.0 g, 98.6 mmol) was dissolved in a solution of sodium hydroxide (15.0 g, 375 mmol, 3.8 eq) in water (37.5 mL). The light brown solution was cooled to 20 °C and treated, with stirring, with 7.75 ml portions of 40% sodium hydroxide and 33.8 ml portions of 10% potassium persulfate solution, starting with the hydroxide, at such a rate that a temperature of 30-35 °C was maintained until ten portions of each had been added. After the addition was complete, stirring was continued for 1 h, the mixture was allowed to stand at room temperature for 16-20 h, and then conical HCl was added until Congo blue. The unreacted 3-methylsalicylic acid separated at this point as a solid and was removed by filtration. The filtrate was extracted with ether several times (5x50 ml) to give a residue of 3-methylsalicylic acid. The aqueous solution was then treated with cone HCl (100 mL) and then refluxed for 2 h to decompose the monosulfate intermediate. The warm solution was allowed to cool to room temperature and the almost black crystalline solid that precipitated was filtered, washed with water and dried. Extraction of the aqueous filtrate with ether gave an additional batch of 2,5-dihydroxy-3-methylbenzoic acid as a brown solid (total: 7.71 g, 47%). Mp 212-214 °C;<1>H nmR (250 MHz, DMSO): δ 10.94 (br, s,, 1H), 7.00 (dd, J = 3.0 and 0.75,<1>Har), 6.86 (dd, J = 3.0 and 0.75,<1>Har), 2.12 (s, 3H).

Esterification. Concentrated sulfuric acid (0.7 mL) was carefully added at 0 °C and under nitrogen to a solution of 2,5-dihydroxy-3-methylbenzoic acid (2.05 g, 12.2 mmol) in MeOH (7 mL). The reaction mixture was then stirred for 6 h at 100 °C. After cooling to room temperature, the solvent was removed under reduced pressure. The resulting crude product was dissolved in ethyl acetate (50 ml) and the solution was washed with water (20 ml), 10% aq. NaHCOs (20 ml), 5% aq. HCl (20 mL) and brine (20 mL), then dried (MgSO4). After filtration, the organic layer was concentrated in vacuo to give a residue which was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate = 9/1) to give methyl 2,5-dihydroxy-3-methylbenzoate as a white solid (370 mg, 75%). Mp 101-103 °C;<1>H nmR (250 MHz, CDCl3): δ 10.56 (d, J = 0.50 Hz , 1H), 7.12 (dd, J = 3.25 and 0.50 Hz ,<1>Har), 6.90 (dt, J = 3.00 and 0.50 Hz ,<1>Har), 4.45 (s, 3H), 2.24 (s, 3H); HRMS (ESI<+>): m/z calcd for C9H11O4 [M+H]<+>: 183.0652; found 183.0651.

Acetylation. Excess acetic anhydride (10 mL) was added to a solution of methyl 2,5-dihydroxy-3-methyl-benzoate (4.43 g, 24.3 mmol) in pyridine (10 mL) under argon. After stirring for 12 h at room temperature, pyridine and acetic anhydride were eliminated by co-evaporation with toluene (25 mL) under reduced pressure. The crude product was then dissolved in ethyl acetate (15 mL) and the solution was washed sequentially with 2% aqueous HCl (5 mL), saturated aqueous NaHCO3 (5 mL), and brine (5 mL). The organic phase was dried (MgSO 4 ) and the solvent was evaporated under reduced pressure. The resulting colorless oil was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate = 8/2) to give the acetylated title product (2,5-acetoxy-3-methyl-benzoate) as a white solid (6.25 g, 97%). Mp 69-71 °C;<1>H nmR (250 MHz, CDCl3): δ 7.58 (dd, J = 3.00 and 0.50 Hz ,<1>Har), 7.18 (dd, J = 3.00 and 0.75 Hz , 1Har), 3.85 (s, 3H), 2.37 (s, 3H), 2.30 (s, 3H), 2.22 (s, 3H); HRMS (ESI<+>): m/z calcd for C13H18NO6 [M+NH4]<+>: 284.1129; found 284.1128.

Methyl 2,5-diacetoxy-3-dibromomethylbenzoate. A solution of methyl 2,5-acetoxy-3-methylbenzoate (2.15 g, 8.08 mmol), N -bromosuccinimide (2.87 mg, 16.2 mmol, 2.0 eq.), and azobisisobutyronitrile (AIBN, 27.0 mg, 0.162 mmol, 0.02 eq.) in carbon tetrachloride (45 mL) was refluxed for 12 h until a white solid formed. floated on the surface. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The resulting colorless oil was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate = 9/1) to give the dibrominated product as a white solid (3.29 g, 84%). Mp 117-119 °C;<1>H nmR (250 MHz, CDCl3): δ 7.86 (d, J = 2.75 Hz ,<1>Har), 7.78 (d, J = 2.75 Hz ,<1>Har), 6.81 (s, 1H), 3.86 (s, 3H), 2.42 (s, 3H), 2.33 (s, 3H); HRMS (ESI<+>): m /z calculated for C13H12Br2NaO6 [M+Na]<+>: 444.8893; found 444.8896.

Methyl 2,5-diacetoxy-3-formylbenzoate. A solution of methyl 2,5-diacetoxy-3-dibromomethylbenzoate (2.30 g, 5.42 mmol) and silver nitrate (2.29 g, 13.5 mmol, 2.5 eq.) in acetone - H2O (4.7:1, 40 ml) was stirred at room temperature in the dark for 12 h. The mixture was then filtered and the filtrate was extracted with ethyl acetate (2 x 20 ml). The combined organic layer was washed with brine (5 mL), dried (MgSO 4 ) and concentrated under reduced pressure. The colorless oil thus obtained was purified by flash chromatography on silica gel (petroleum ether/ethyl acetate = 9/1) to give the aldehyde as a yellow solid (1.14 g, 75%). Mp 102-104 °C; 1H nmR (250 MHz, CDCl3): □ 10.17 (s, 1H), 8.00 (d, J = 3.00 Hz , 1Har), 7.82 (d, J = 3.00 Hz , 1Har), 3.90 (s, 3H), 2.44 (s, 3H), 2.34 (s, 3H).

Monomethyl ester of 2,5-diacetoxyisophthalic acid (KI-2Ac2). A solution of sodium hydrogen phosphate (1.35 g, 9.81 mmol, 2.5 eq) in water (3.5 mL) was added dropwise to a solution of methyl 2,5-diacetoxy-3-formylbenzoate (1.10 g, 3.93 mmol) in DMSO (14 mL). The mixture was then cooled to 0 °C and a solution of sodium chlorite (1.06 g, 9.34 mmol, 2.4 eq) in water (3.5 mL) was slowly added. After stirring at room temperature for 72 h, the mixture was quenched with saturated aqueous NaHCO 3 solution (10 mL) and extracted with ethyl acetate (2 x 30 mL). The aqueous phase was then acidified with 1M HCl to pH = 1 and extracted with ethyl acetate (2 x 30 ml).

The combined organic layer was washed with brine (5 mL), dried (MgSO 4 ) and concentrated under reduced pressure. The resulting orange oil was used in the next step without any purification.

Synthesis of monoamides

Scheme 10. Synthesis of monoamide IIa

Protocols: Synthesis of monoamides

Amide Coupling and Deprotection Procedures

See General procedures from KI-1 and KI-6 steps [3], [4], [5], same procedures from KI-2, KI-2Ac2 and KI-7

Examples

For conditions and yields: See Table 2 (Figures 5A-C)

33) 3-(2-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound IIa-001a:

UPLC-MS (acid procedure, 2 min): rt = 0.86 min; m/z = 318.0 [M+H]<+>, peak area >92%

<1>H nmR (400 MHz, DMSO-d6) δ 13.49 (s, 1H), 12.16 (s, 1H), 9.54 (s, 1H), 8.70 (dd, J = 8.5, 1.2 Hz , 1H), 7.99 (dd, J = 7.9, 1.7 Hz , 1H), 7.70 - 7.51 (m, 2H), 7.42 (d, J = 3.3 Hz , 1H), 7.20 (td, J = 7.6, 1.2 Hz , 1H).

HRMS: [M+H]<+>, calcd. for C15H12NO7: 318.06083, found: 318.06082

Biological data: IIa-001a: FGF-1 IC50 [µM] = 8.6; FGF-2 IC50 [µM] = 11; VEGF-A1 IC50 [µM] = 150; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 30.5; PMN ROS inhibition IC50 [µM] = 0.408; Inhibition of neutrophil adhesion [%] = 36.24

34) 3-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound IIa-001aTz:

UPLC-MS (acid procedure, 2 min): rt = 0.71 min; m/z = 342.1 [M+H]<+>, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 9.49 (s, 1H), 8.51 (d, J = 8.3 Hz , 1H), 7.87 (d, J = 7.7 Hz , 1H), 7.72 - 7.51 (m, 2H), 7.51 - 7.26 (m, 2H)

HRMS: [M+H]<+>, calcd. for C15H12N5O5: 342.08329, found: 342.08317

Biological data: IIa-001a-Tz: FGF-1 IC50 [µM] = N.D.; FGF-2 IC50 [µM] = 9.8; VEGF-A1 IC50 [µM] = 187; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 74.15; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 24.18

Ethyl ester

UPLC-MS (acid procedure, 4 min): rt = 1.46 min; m/z = 370.1 [M+H]<+>, peak area >92%

<1>H nmR (400 MHz, DMSO-d6) δ 11.51 (s, 1H), 11.37 (s, 1H), 9.63 (s, 1H), 8.47 (d, J = 8.3 Hz , 1H), 7.92 (dd, J = 7.8, 1.6 Hz , 1H), 7.66 - 7.57 (m, 2H), 7.43 (d, J = 3.2 Hz , 1H), 7.38 (td, J = 7.6, 1.2 Hz , 1H), 4.40 (q, J = 7.1 Hz , 2H), 1.36 (t, J = 7.1 Hz , 3H).

HRMS: [M+H]<+>, calcd. for C17H16N5O5: 370.11460, found: 370.11444

Biological data: IIa-001aTz-E1: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 42; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 68.86; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 81.94

35) 2,5-Dihydroxy-3-(2-sulfophenylaminocarbonyl)benzoic acid, compound IIa-001c:

UPLC-MS (acid procedure, 2 min): rt = 0.63 min; m/z = 354.0 [M+H]<+>, peak area >80%

<1>H nmR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 9.48 (s, 1H), 8.34 - 8.27 (m, 1H), 7.72 (dd, J = 7.7, 1.7 Hz , 1H), 7.52 (d, J = 3.2 Hz , 1H), 7.41 - 7.31 (m, 2H), 7.08 (td, J = 7.5, 1.2 Hz , 1H).

HRMS: [M+H]<+>, calcd. for C14H12NO8S: 354.02781, found: 354.02781

Biological data: IIa-001c: FGF-1 IC50 [µM] = N.D.; FGF-2 IC50 [µM] = 71; VEGF-A1 IC50 [µM] =283; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 72.62; PMN ROS inhibition IC50 [µM] = N.D.;

Inhibition of neutrophil adhesion [%] = 43.53

36) 3-(3-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound IIa-002a:

<1>H nmR (250 MHz, CD3OD): δ 8.37 (t, 1H), 7.93 (br d, 1H), 7.81 (br d, 1), 7.76 (d, 1H), 7.52 (d, 1H), 7.48 (t, 1H).

HRMS (ESI-): [MH]-, calcd. for C15H10NO7: 316.0461, found:

318.0463

Biological data: IIa-002a: FGF-1 IC50 [µM] = 19; FGF-2 IC50 [µM] = 131; VEGF-A1 IC50 [µM] = 150; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 43.3; PMN ROS inhibition IC50 [µM] = 0.299; Inhibition of neutrophil adhesion [%] = 17.39

37) 3-(4-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid)amide, compound IIa-003a:

<1>H nmR (250 MHz, CD3OD): □8.06 (BB' of AA'BB', 2H), 7.85 (AA' of AA'BB', 2H), 7.75 (d, 1H), 7.58 (d, 1H)

HRMS (ESI-): calcd for C15H10NO7 [MH]-: 316.0461; found 316.0462

Biological data: IIa-003a: FGF-1 IC50 [µM] = 22; FGF-2 IC50 [µM] = 13; VEGF-A1 IC50 [µM] = 150; VEGFR-inhibition of phosphorylation IC50 [µM] =2.5; PMN ROS [inhibition at 0.3 µm [%] = 59.3; PMN ROS inhibition IC50 [µM] = N.D.;

Inhibition of neutrophil adhesion [%] = 35.27

38) 3-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound IIa-004a:

UPLC-MS (acid procedure, 2 min): rt = 0.76 min; m/z = 334.0 [M+H]<+>, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 14.02 (brs, 1H), 11.07 (brs, 1H), 10.32 (s, 1H), 9.47 (brs, 1H), 8.27 (d, J = 2.7 Hz , 1H), 7.76 (dd, J = 8.9, 2.7 Hz, 1H), 7.42 (d, J = 3.2 Hz , 1H), 7.36 (d, J = 3.2 Hz , 1H), 6.96 (d, J = 8.9 Hz , 1H).

HRMS: [M+H]<+>, calcd. for C15H12NO8: 334.05574, found: 334.05571

Biological data: IIa-004a: FGF-1 IC50 [µM] = 47; FGF-2 IC50 [µM] = 33; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 63.5; PMN ROS inhibition IC50 [µM] = 1.002;

Inhibition of neutrophil adhesion [%] = 36

39) 3-(2-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound IIa-006a:

UPLC-MS (acid procedure, 2 min): rt = 0.65 min; m/z = 334.0 [M+H]<+>, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 13.36 (s, 1H), 11.83 (s, 1H), 9.62 (s, 1H), 9.47 (s, 1H), 8.47 (d, J = 9.1 Hz , 1H), 7.64 (d, J = 3.3 Hz , 1H), 7.41 (d, J = 3.2 Hz , 1H), 7.37 (d, J = 3.0 Hz , 1H), 7.03 (dd, J = 9.1, 3.0 Hz , 1H).

HRMS: [M+H]<+>, calcd. for C15H12NO8: 334.05574, found: 334.05548

Biological data: IIa-006a: FGF-1 IC50 [µM] =N.D.; FGF-2 IC50 [µM] = 43; VEGF-A1 IC50 [µM] = 121; VEGFR-inhibition of phosphorylation IC50 [µM] =2.9; PMN ROS [inhibition at 0.3 µm [%] = 84.29; PMN ROS inhibition IC50 [µM] = N.D.;

Inhibition of neutrophil adhesion [%] = 20.29

40) 3-(3-carboxy-2,5-dihydroxybenzamido)phthalic acid, compound IIa-011a:

UPLC-MS (acid procedure, 2 min): rt = 0.72 min; m/z = 360.1 [M-H]-, peak area >77%

<1>H nmR (400 MHz, DMSO-d6) δ 10.83 (s, 1H), 9.55 (s, 1H), 8.43 (dd, J = 7.9, 1.5 Hz , 1H), 7.73 (d, J = 3.3 Hz , 1H), 7.63 - 7.53 (m, 2H), 7.46 (d, J = 3.3 Hz, 1H).

HRMS: [M+H]<+>, calcd. for C16H12NO9: 362.05066, found: 362.05045,

Biological data: IIa-011a: FGF-1 IC50 [µM] = 141; FGF-2 IC50 [µM] = 123; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 58.1; PMN ROS inhibition IC50 [µM] = 1.18; Inhibition of neutrophil adhesion [%] = 26

41) 2-(3-carboxy-2,5-dihydroxybenzamido)terephthalic acid, compound IIa-012a:

UPLC-MS (acid procedure, 2 min): rt = 0.73 min; m/z = 362.0 [M+H], peak area >97%

<1>H nmR (DMSO-d6) δ: 13.81 (s, 1H), 13.34 (s, 1H), 12.23 (s, 1H), 9.50 (s, 1H), 9.30 (d, J = 1.6 Hz , 1H), 8.06 (d, J = 8.2 Hz , 1H), 7.71 (dd, J = 8.2, 1.7 Hz , 1H), 7.64 (d, J = 3.3 Hz , 1H), 7.43 (d, J = 3.3 Hz , 1H).

HRMS: [M+H]<+>, calcd. for C16H12NO9: 362.05066, found: 362.05046,

Biological data: IIa-012a: FGF-1 IC50 [µM] = 150; FGF-2 IC50 [µM] = 150; VEGF-A1 IC50 [µM] = 32; VEGFR-inhibition of phosphorylation IC50 [µM] =3.31; PMN ROS [inhibition at 0.3 µm [%] = 44.8; PMN ROS inhibition IC50 [µM] = 0.313; Inhibition of neutrophil adhesion [%] = 7.5

42) 2-(3-carboxy-2,5-dihydroxybenzamido)isophthalic acid, compound IIa-013a:

UPLC-MS (acid procedure, 2 min): rt = 1.68 min; m/z = 362.0 [M+H]<+>, peak area >96%

<1>H nmR (400 MHz, DMSO-d6) δ 13.12 (brs, 3H), 11.71 (s, 1H), 9.47 (s, 1H), 7.96 (s, 1H), 7.94 (s, 1H), 7.66 (d, J = 3.3 Hz), 7.44 (d, J = 3.3 Hz). , 1H), 7.35 (t, J = 7.8 Hz , 1H).

HRMS: [M+H]<+>, calcd. for C16H12NO9: 362.05066, found: 362.05047

Biological data: IIa-013a: FGF-1 IC50 [µM] = 137; FGF-2 IC50 [µM] = 12; VEGF-A1 IC50 [µM] = 34; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 57.68; PMN ROS inhibition IC50 [µM] = 2.54;

Inhibition of neutrophil adhesion [%] = 15.75

43) 4-(3-carboxy-2,5-dihydroxybenzamido)phthalic acid, compound IIa-014a:

UPLC-MS (acid procedure, 2 min): rt = 0.63 min; m/z = 362.1 [M+H]<+>, peak area >88%

<1>H nmR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 9.45 (s, 1H), 8.01 (d, J = 2.2 Hz , 1H), 7.86 (dd, J = 8.5, 2.2 Hz , 1H), 7.74 (d, J = 8.5 Hz , 1H), 7.45 -7.33 (m, 2H).

HRMS: [M+H]<+>, calcd. for C16H12NO9: 362.05066, found: 362.05048

Biological data: IIa-014a: FGF-1 IC50 [µM] = 40; FGF-2 IC50 [µM] = 61; VEGF-A1 IC50 [µM] = 91; VEGFR-inhibition of phosphorylation IC50 [µM] =17.6; PMN ROS [inhibition at 0.3 µm [%] = 73.94; PMN ROS inhibition IC50 [µM] = N.D.;

Inhibition of neutrophil adhesion [%] = 21.5

44) 5-(3-carboxy-2,5-dihydroxybenzamido)isophthalic acid, compound IIa-015a:

<1>H nmR (250 MHz, CD3OD): δ 8.60 (d, J = 1.5 Hz , 2H), 8.44 (t, J = 1.5 Hz , 1H), 7.75 (d, J = 3.2 Hz , 1H), 7.56 (d, J = 3.2 Hz , 1H)

HRMS (ESI+): m/z calcd for C16H12NO9 [M+H]+: 362.0507; found 362.0505 [LM-163]

Biological data: IIa-015a: FGF-1 IC50 [µM] = N.D.; FGF-2 IC50 [µM] = 125; VEGF-A1 IC50 [µM] = N.D.; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = ND; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = ND

45) (3-(3-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound IIa-033a:

UPLC-MS (acid procedure, 2 min): rt = 0.78 min; m/z = 330.1 [M-H]-, peak area >98%

<1>H nmR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.45 (s, 1H), 7.66 (t, J = 1.9 Hz , 1H), 7.63 - 7.57 (m, 1H), 7.45 (d, J = 3.3 Hz , 1H), 7.38 (d, J = 3.2 Hz , 1H), 7.29 (t, J = 7.8 Hz , 1H), 7.05 - 6.95 (m, 1H), 3.56 (s, 2H).

HRMS: [M+H]<+>, calcd. for C16H14NO7: 332.07648, found: 332.07644,

Biological data: IIa-033a: FGF-1 IC50 [µM] = 35; FGF-2 IC50 [µM] = 32; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =4.9; PMN ROS [inhibition at 0.3 µm [%] = 50.64; PMN ROS inhibition IC50 [µM] = 0.914; Inhibition of neutrophil adhesion [%] = 11.75

46) 3-(2-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound IIa-034a:

UPLC-MS (acid procedure, 2 min): rt = 0.74 min; m/z = 332.0 [M+H]<+>, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 12.48 (s, 1H), 10.65 (s, 1H), 9.30 (s, 1H), 7.97 (d, J = 8.3 Hz , 1H), 7.64 (d, J = 3.2 Hz , 1H), 7.40 (d, J = 3.3 Hz , 1H). 1H), 7.33 - 7.26 (m, 2H), 7.13 (td, J = 7.5, 1.3 Hz , 1H), 3.70 (s, 2H).

HRMS: [M+H]<+>, calcd. for C16H14NO7: 332.07648, found: 332.07653 Biological data: IIa-034a: FGF-1 IC50 [µM] = 22; FGF-2 IC50 [µM] = 5.7; VEGF-A1 IC50 [µM] = 86; VEGFR-inhibition of phosphorylation IC50 [µM] =100; PMN ROS [inhibition at 0.3 µm [%] = 69.2; PMN ROS inhibition IC50 [µM] = N.D.;

Inhibition of neutrophil adhesion [%] = 26.58

Diethyl ester

UPLC-MS (acid procedure, 2 min): rt = 1.12 min; m/z = 388.1 [M+H]<+>, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 9.58 (s, 1H), 7.77 (t, J = 8.5 Hz , 1H), 7.65 (d, J = 3.4 Hz , 1H), 7.42 (d, J = 3.4 Hz , 1H), 7.36 - 7.31 (m, 2H), 7.20 (t, J = 7.4 Hz , 1H), 4.39 (q, J = 7.1 Hz , 2H), 4.05 (q, J = 7.1 Hz , 2H), 3.77 (s, 2H), 1.36 (t, J = 7.1 Hz , 3H), 1.13 (t, J = 7.1 Hz , 3H).

HRMS: [M+H]<+>, calcd. for C20H22N07: 388.13907, found: 388.13887

Biological data: IIa-034a-E2: FGF-1 IC50 [µM] =N.D.; FGF-2 IC50 [µM] = 164; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 68.73; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 11.86

47) 3-(3,4-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound IIa-035a:

UPLC-MS (acid procedure, 2 min): rt = 0.69 min; m/z = 318.0 [M-H]-, peak area >99%

<1>H nmR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.84 (s, 1H), 8.75 (s, 1H), 7.55 (d, J = 3.2 Hz , 1H), 7.34 (d, J = 3.2 Hz , 1H), 6.74 (d, J = 2.1 Hz , 1H). 6.67 (d, J = 8.0 Hz , 1H), 6.58 (dd, J = 8.1, 2.1 Hz , 1H), 4.34 (d, J = 5.7 Hz , 2H).

HRMS: [M+H]<+>, calcd. for C15H14NO7: 320.07647, found: 320.07625.

Biological data: IIa-035a: FGF-1 IC50 [µM] = 16; FGF-2 IC50 [µM] = 61; VEGF-A1 IC50 [µM] = 45; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.52; PMN ROS [inhibition at 0.3 µm [%] = 76.99; PMN ROS inhibition IC50 [µM] = 1;

Inhibition of neutrophil adhesion [%] = 36.67

48) 3-(2-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid, compound IIa-053a:

UPLC-MS (acid procedure, 4 min): rt = 1.08 min; m/z = 332.0 [M+H]<+>, peak area >97%

<1>H nmR (DMSO-d6) δ: 13.13 (brs, 1H), 12.73 (brs, 1H), 9.40 (s, 1H), 9.05 (s, 1H), 7.91 (dd, J = 7.8, 1.4 Hz , 1H), 7.65 - 7.29 (m, 5H), 4.80 (d, J = 6.1 Hz, 2H).

HRMS: [M+H]<+>, calcd. for C16H14NO7: 332.07648, found: 332.07626

Biological data: IIa-053a: FGF-1 IC50 [µM] = 84; FGF-2 IC50 [µM] = 18; VEGF-A1 IC50 [µM] = 37; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.27; PMN ROS [inhibition at 0.3 µm [%] = 63.13; PMN ROS inhibition IC50 [µM] = 0.62;

Inhibition of neutrophil adhesion [%] = 20; Whole blood: GM-CSF IC50 [µM] = >100; IFNi IC50 [µM] = 1.04; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 1.67; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 2.2; IL-9 IC50 [µM] = 1.41; IL-10 IC50 [µM] = >100; IL-12p70 IC50 [µM] = 0.27; IL-13 IC50 [µM] = 29.9; IL-17A IC50 [µM] = >100; IL-17F IC50 [µM] = 28.8; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = >100; IL-33 IC50 [µM] = 12.1; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.02; TNF β IC50 [µM] = 91.5.

Example 1.6. Type IIb molecules: Diamides from diamines, 1 example

Scheme 11. Synthesis of type IIb diamides

Procedure: see general procedure from KI-1 [3], [4], [5]

For example:

For conditions and yields: See Table 2 (Figures 5A-C)

49) 3,5-bis(2,5-dihydroxy-3-carboxybenzoylamino)benzoic acid, compound IIb-010a:

UPLC-MS (acid procedure, 4 min): rt = 0.98 min; m/z = 511.1 [M-H]-, peak area >93%

<1>H nmR (400 MHz, DMSO-d6) δ 10.81 (s, 2H), 10.65 (s, 2H), 8.38 (m, 1H), 8.12 (d, J = 2.0 Hz , 2H), 7.39 (d, J = 2.3 Hz , 4H).

HRMS: [M+H]<+>, calcd. for C23H17N2O12: 513.07760, found:

513.07774

Biological data: IIb-010a: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 102; VEGF-A1 IC50 [µM] = 28; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 81.87; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 59.84

Example 1.7. Type IIc molecules: Diamides from diacids, 2 examples

Scheme 12. Synthesis of type IIc amides with R = R'

Procedure: see general procedure from KI-6 [3], [4], [5]

For examples

For conditions and yields: See Table 2 (Figures 5A-C)

50) 5-(3-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid, compound IIc-007a:

UPLC-MS (acid procedure, 4 min): rt = 1.20 min; m/z = 469.1 [M+H]<+>, peak area >91%

<1>H nmR (400 MHz, DMSO-d6) δ 10.41 (s, 2H), 9.51 (s, 1H), 8.22 (d, J = 2.7 Hz , 2H), 7.79 (dd, J = 9.0, 2.7 Hz , 2H), 7.58 (s, 2H), 6.98 (d, J = 8.9 Hz) , 2H).

<13>C nmR (100 MHz, DMSO-d6) δ 172.1, 166.1, 158.3, 152.0, 149.4, 130.2, 129.5 (CH), 122.8 (CH), 120.3, 119.8 (CH), 117.7 (CH), 113.0.

HRMS: [M+H]<+>, calcd. for C22H17N2O10: 469.08777, found: 469.08803

Biological data: IIc-007a: FGF-1 IC50 [µM] = 11; FGF-2 IC50 [µM] = 91; VEGF-A1 IC50 [µM] = 8.2; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.44; PMN ROS [inhibition at 0.3 µm [%] = 93.04; PMN ROS inhibition IC50 [µM] =ND;

Inhibition of neutrophil adhesion [%] = 64.61; Whole blood: GM-CSF IC50 [µM] = 26; IFNγ IC50 [µM] = 0.11; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 12.8; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.05; IL-9 IC50 [µM] = 2.14; IL-10 IC50 [µM] = 17.9; IL-12p70 IC50 [µM] = 3.37; IL-13 IC50 [µM] = 0.25; IL-17A IC50 [µM] = 0.01; IL-17F IC50 [µM] = 0.26; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = 9.41; IL-33 IC50 [µM] = 0.18; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.03; TNF β IC50 [µM] = 0.29

Alternative large-scale synthesis of IIc-007a

2,5-dibenzyloxyisophthalic acid (KI-7)

2,5-dibenzyloxyisophthalaldehyde (Scheme 9b) (300 g, 866 mmol), resorcinol (286 g, 2.60 mol), KH2PO4 (354 g, 2.60 mol), acetone (1.5 mL), and water (516 mL) were added to a 5 L jacketed vessel. The suspension was stirred at 15-25 °C. A solution of 80% NaClO2 (294 g, 2.60 mol) in water (1 L) was heated at 15-30 °C for 90 min (exothermic). After the addition was complete, the reaction was stirred at 15-25 °C for 1 hour. A solution of 85% H3PO4(85 mL, 1.24 mol) in water (1175 mL) [~1M] was charged over 10 min at T<30 °C (exothermic) to give a precipitate. The batch was cooled to 0-5 °C and filtered. The solids were washed with water (3x12 L) and dried in an oven (50 °C) to give 325.6 g of diacid KI-7. HPLC:

98.9%; nmR >95%. Adjusted yield (90.6%).

5-(3-(3-Methoxycarbonyl-4-hydroxyphenylaminocarbonyl)-2,5-dibenzyloxybenzamido)-2-hydroxybenzoic acid methyl ester. KI-7 diacid (80 g, 211 mmol), HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (176.8 g, 466 mmol) and THF (560 ml) were poured into a 2 L jacketed vessel. The batch was stirred at 15-25 °C and N-methylimidazole (50.6 mL, 635 mmol) was added. After 30 min, methyl 5-amino-2-hydroxybenzoate (77.8 g, 466 mmol) was added and the reaction was stirred at 25 °C overnight. Water (1.2 L) was added and the batch was stirred at 20 °C for 30 min. The batch was filtered, washed with water

(2x480 ml), then MeCN (320 ml). The resulting solid (236 g) was charged back into the vessel along with MeCN (800 mL). The suspension was heated to 50 °C for 40 min and then cooled to 20 °C. The batch was filtered and washed with MeCN (320 mL). nmR analysis of the solid (156 g) showed no TMU, HOBt, nmI or HBTU. The material was dried at 50 °C overnight to give 125 g of diamide. HPLC: 98.2%. nmR: >97%. Yield: 88%.

5-(3-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dibenzyloxybenzamido)-2-hydroxybenzoic acid. A 2 L jacketed vessel was charged with the previous diamide (110 g, 163 mmol), THF (550 mL) and water (1100 mL). The batch was stirred at 15-25 °C and charged with 85% KOH (32.2 g, 488 mmol) (slight exothermic). The solution was heated to 50 °C for 4 h, then cooled to 20 °C and stirred overnight. 6 M aq. AcOH (550 mL, 3.3 mol) was then charged over 30 min at 15-25 °C. After the addition was complete, the batch was stirred for 30 minutes and then filtered. The solids were washed with water (3x550 ml) and then dried in an oven at 50 °C. This gave 102 g of the free diacid. HPLC:

98.9% (0.3% mono-amide, 0.19% monoacid). nmR: >97%. Yield: 97%.

5-(3-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid IIc-007a. 10% Pd/C (10 g, 50% wet, type 87L) was added to a 2 L jacketed vessel under N 2 , followed by the previous diacid (100 g, 154 mmol), AcOH (5 ml, 88 mmol) and THF (1200 ml). The batch was mixed and then sparged with H 2 and heated at 40 °C for 2 hours.

The batch was sparged with N2 and then filtered (GF/F). The vessel was washed with THF (300 mL) and the washings were used to wash the catalyst on the filter. The filtrate was loaded into a vessel and the volume was adjusted to 2 L with THF (400 mL). The solution was heated to 30 °C, SPM32 (10 g) was added, and the batch was stirred at 30 °C overnight. The SPM32 was filtered and washed with THF (200 mL). The solvent was removed in vacuo to give a light yellow solid (110 g). nmR analysis showed 28% THF. The material was suspended in EtOH (1 L) at 20 °C for 2 h and then filtered. The solids were washed with EtOH (400 mL) and dried in an oven at 60 °C overnight. nmR showed 8.7% EtOH, no THF. The material was further dried at 80 °C for 4 nights to give 66.5 g of IIc-007a as an off-white solid in 92% yield. HPLC: 99.5% (0.31% monoamide). nmR: 4.6% EtOH, no THF. Pd by ICP-OES: <2 ppm.

IIc-007a-THF solvate: Concentration of the THF filtrate gave a yellow solid, typically containing 25-30% THF by nmR, which could not be removed by drying, indicating a non-stoichiometric solvate. A small sample of the THF solvate (1.45 g) was heated at 50 °C in THF (10 ml) for 1 hour, cooled to room temperature and isolated, washed with 3 ml of THF. This gave 1.13 g of IIc-007a-THF. nmR: 27% THF. HPLC: 99.6% (0.1% monoamide). Isolation of the THF solvate by filtration leads to an increase in purity and efficient cleaning of the main impurity (monoamide, IIa-004a) from 0.7% to 0.1%. Recovery yield: 78%. THF solvate solubility in THF calculated to be ~25 mg/ml at 20 °C.

Desolvation tests were performed on the material in acetone, EtOH and EtOAc. Each batch was heated for 1 hour at 50 °C in 20 vol. solvent, then isolated at room temperature and dried in an oven (60 °C). Ethanol was chosen as the desolvation solvent because of the low level of EtOH incorporated into the product after drying and the excellent scavenging of THF from the system. The desolvation step was performed on 12.1 g of product (25% THF). EtOH (20 vol.) was charged to the material and the batch was stirred at RT overnight. The sample was filtered and this indicated successful desolvation at room temperature. Total yield: 8.67 g. HPLC: 99.4%. nmR: >97% (0.8% EtOH). XRPD showed a high degree of crystallinity of the isolated product.

Product IIc-007a also forms DMSO solvates (DMSO:product ratio 3:2).

51) 2-(3-(2-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-5-hydroxylbenzoic acid, compound IIc-009a:

UPLC-MS (acid procedure, 2 min): rt = 0.79 min; m/z = 469.1 [M+H]<+>, peak area >95%

<1>H nmR (400 MHz, DMF-d7) δ 10.11 (s,1H), 10.01 (s,2H), 8.71 (d, J = 9.0 Hz , 2H), 8.01 (s, 2H), 7.82 (d, J = 3.0 Hz , 2H), 7.46 (d, J = 8.9 Hz , 2H).

HRMS: [M+H]<+>, calcd. for C22H17N2O10: 469.08777, found: 469.08714

Biological data: IIc-009a: FGF-1 IC50 [µM] = 30; FGF-2 IC50 [µM] = N.D.; VEGF-A1 IC50 [µM] = 182; VEGFR-inhibition of phosphorylation IC50 [µM] =1.7; PMN ROS [inhibition at 0.3 µm [%] = 75.31; PMN ROS inhibition IC50 [µM] =ND; Inhibition of neutrophil adhesion [%] = 53.15; Whole blood: GM-CSF IC50 [µM] = >100; IFNγ IC50 [µM] = 0.54; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 19.6; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.27; IL-9 IC50 [µM] = 18.9; IL-10 IC50 [µM] = 11.31; IL-12p70 IC50 [µM] = >100; IL-13 IC50 [µM] = 0.13; IL-17A IC50 [µM] = 0.001; IL-17F IC50 [µM] = 0.13; IL-18 IC50 [µM] = 2.95; IL-21 IC50 [µM] = 8.01; IL-33 IC50 [µM] = 0.29; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.11; TNF β IC50 [µM] = 6.94.

Example 1.8: molecules of type Illa: Monoamides, 4 examples

Synthesis of precursors)

Scheme 13. Synthetic intermediates: KI-8 and KI-9 from KI-1

Synthetic protocols

Intermediates KI-8 and KI-9 (via KI-10 and KI-11)

Ethyl 2,5-bis(benzyloxy)-4-(hydroxymethyl)benzoate (KI-10) [Step 1]: To a cooled -20 °C solution of HI-1 (62.3 g, 153 mmol) in THF (700 ml) under a positive flow of inert gas (N2) was slowly added a pre-cooled solution of BH3.THF (615.0 ml, 615 mmol) (at -10 °C). The addition was done in such a way that the internal temperature of the reaction never exceeded -15 °C. The reaction is slightly heated, the internal temperature is not allowed to exceed -7 °C. The mixture was maintained at this temperature for 2.5 h. The reaction mixture was then poured into ice/water (8 L). The resulting cloudy white mixtures were allowed to stir at room temperature for 2 h, and the residual white suspension was filtered through a funnel (Porosity 3). The white solid thus obtained was further dried in a vacuum oven at 40 °C overnight to give ethyl 2,5-bis(benzyloxy)-4-(hydroxymethyl)benzoate (HI-10) (60.5 g, 97% yield) as a white solid.

UPLC-MS (acid procedure, 2 min): rt = 1.25 min; m/z = 391.2 [M-H]-, peak area >68%

<1>H nmR (400 MHz, DMSO-d6) δ 7.54 - 7.48 (m, 2H), 7.48 - 7.43 (m, 2H), 7.43 - 7.35 (m, 4H), 7.35 - 7.27 (m, 4H), 5.28 (t, J = 5.4 Hz, 1H), 5.13 (s, 2H), 5.11 (s, 2H), 4.58 (d, J = 5.4 Hz , 2H), 4.25 (q, J = 7.1 Hz , 2H), 1.26 (t, J =

7.1 Hz, 3H).

Alternatively, intermediate KI-10 can be obtained from KI-1 via mixed anhydride (treatment of KI-1 with isobutylchloroformate in THF in the presence of triethylamine) followed by reduction of the mixed anhydride with sodium borohydride in THF (85-90% yields, 150 g-scale).

Ethyl 2,5-bis(benzyloxy)-4-(chloromethyl)benzoate (KI-11) [Step 2]: Tosyl chloride (17.0 g, 70 mmol) was slowly added to a cooled solution (ice bath) of KI-10 (25.0 g, 64 mmol), DIPEA (12.2 mL, 70 mmol), and DMAP (0.778 g, 6 mmol) in DCM (260 mL). The reaction mixture was stirred at 60 °C for 30 h, after which the reaction was judged to be complete. DCM (100 mL) was added and the organic layer was separated, washed with saturated aqueous sodium bicarbonate (4 x 50 mL), water (2 x 50 mL), and brine (50 mL), dried over sodium sulfate, and concentrated. The crude product was subjected to column chromatography (Hexane/EtOAc 0-20%) to give ethyl 2,5-bis(benzyloxy)-4-(chloromethyl)benzoate (KI-11) (23.5 g, 75%).

UPLC-MS (acid procedure, 2 min): rt = 1.39 min; m/z = no ions, peak area >84%.

<1>H nmR (400 MHz, DMSO-d6) δ 7.54 - 7.44 (m, 4H), 7.43 - 7.36 (m, 6H), 7.36 - 7.28 (m, 2H), 5.18 (s, 2H), 5.13 (s, 2H), 4.76 (s, 2H), 4.26 (q, J = 7.1 Hz , 2H), 1.25 (t, J = 7.1 Hz , 3H).

Alternatively, intermediate KI-11 can be obtained by treating KI-10 with thionyl chloride (1.14 eq) in dichloromethane at -10 °C, followed by evaporation of the solvent and precipitation from heptane (90% yield, 300 g scale)

Ethyl 2,5-bis(benzyloxy)-4-(cyanomethyl)benzoate [Step 3]: A suspension of KI-11 (7.5 g, 18.2 mmol) in a mixture of EtOH (90 mL) and H 2 O (45 mL) was treated with potassium cyanide (1.8 g, 27.4 mmol). The reaction mixture was heated to 75 °C and stirred at this temperature for 16 h. The reaction mixture was diluted with water (500 ml) and extracted with EtOAc (2 x 200 ml), the combined organic phases were then washed with brine (200 ml), dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was isolated as a mixture of ethyl 2,5-bis(benzyloxy)-4-(cyanomethyl)benzoate and 2,5-bis(benzyloxy)-4-(cyanomethyl)benzoic acid (7.1 g), the product was used in the next step without further purification.

UPLC-MS (acid procedure, 2 min): rt = 1.30 min; m/z = 402.2 [M+H]<+>, peak area >65% and rt = 1.15 min; m/z = 374.1 [M+H]<+>, peak area >8%.

<1>H nmR (400 MHz, DMSO-d6) δ 7.56 - 7.27 (m, 12H), 5.19 (s, 2H), 5.14 (s, 2H), 4.26 (q, J = 7.1 Hz , 2H), 3.95 (s, 2H), 1.25 (t, J = 7.1 Hz , 3H).

2,5-bis(benzyloxy)-4-(carboxymethyl)benzoic acid (KI-9) [Step 4]: To a suspension of ethyl 2,5-bis(benzyloxy)-4-(cyanomethyl)benzoate (7.1 g, 13.2 mmol) in EtOH (20 mL) was added a solution of sodium hydroxide (8.5 g, 212.5 mmol) in water (50 mL) and the reaction mixture was stirred under reflux. 18 h. The reaction mixture was diluted with water (100 ml) and the resulting solution was washed with EtOAc (2 x 100 ml), the resulting organic layer was extracted with water (100 ml) and all aqueous layers were collected. Then the aqueous layer was acidified to pH ∼ 3 with a saturated aqueous solution of citric acid, the formed precipitate was filtered and dried under reduced pressure. The solid was further dried in a vacuum oven at 40 °C overnight to give 2,5-bis(benzyloxy)-4-(carboxymethyl)benzoic acid (KI-9) (6.4 g, 92% yield) as a yellowish solid.

UPLC-MS (acid procedure, 2 min): rt = 1.07 min; m/z = 393.2 [M+H]<+>, peak area >74%.

<1>H nmR (400 MHz, DMSO-d6) δ 12.46 (s, 2H), 7.55 - 7.27 (m, 11H), 7.19 (s, 1H), 5.11 (s, 2H), 5.09 (s, 2H), 3.61 (s, 2H).

2,5-bis(benzyloxy)-4-(2-ethoxycarbonylmethyl)benzoic acid (KI-8) [Step 5]: KI-9 (5.5 g, 14.0 mmol) was suspended in EtOH (30 mL) and the suspension was treated with thionyl chloride (0.52 mL, 7.1 mmol) and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was then poured into saturated sodium bicarbonate solution (1 L), resulting in a white suspension. The solid was isolated by filtration and further triturated with Et2O (2 × 20 mL). The solid was further dried in a vacuum oven overnight to give 2,5-bis(benzyloxy)-4-(2-ethoxycarbonylmethyl)benzoic acid (KI-8) (4.2 g, 64%) as a white solid.

UPLC-MS (acid procedure, 2 min): rt = 1.23 min; m/z = 419.3 [M-H]-, peak area >79%.

<1>H nmR (400 MHz, DMSO-d6) δ 12.68 (brs, 1H), 7.58 - 7.27 (m, 11H), 7.19 (s, 1H), 5.11 (s, 2H), 5.08 (s, 2H), 4.01 (q, J = 7.1 Hz , 2H), 3.66 (s, 2H), 1.11 (t, J = 7.1 Hz, 3H).

Synthesis of monoamides

Scheme 14. Synthesis of monoamide IIIa

Synthetic protocols

Amide Coupling and Deprotection Procedures:

See general procedures from steps KI-1 and KI-6 [3], [4], [5]

Examples

For terms and yields: See Table 3 (Figure 6)

52) 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)benzoic acid, compound IIIa-001a:

UPLC-MS (acid procedure, 2 min): rt = 0.89 min; m/z = 332.1 [M+H]<+>, peak area >98%

<1>H nmR (400 MHz, DMSO-d6) δ 13.38 (s, 1H), 12.17 (s, 1H), 10.67 (s, 1H), 9.23 (s, 1H), 8.64 (dd, J = 8.5, 1.2 Hz , 1H), 8.00 (dd, J = 7.9, 1.7 Hz, 1H). 1H), 7.62 (ddd, J = 8.7, 7.3, 1.7 Hz , 1H), 7.33 (s, 1H), 7.19 (td, J = 7.6, 1.2 Hz , 1H), 6.80 (s, 1H), 3.49 (s, 2H).

HRMS: [M+H]<+>, calcd. for C16H14NO7: 332.07648, found: 332.07666 Biological data: IIIa-001a: FGF-1 IC50 [µM] = 62; FGF-2 IC50 [µM] = 10; VEGF-A1 IC50 [µM] = 32; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 74.79; PMN ROS inhibition IC50 [µM] = N.D.;

Inhibition of neutrophil adhesion [%] = 30.4

Ethyl methyl ester

UPLC-MS (acid procedure, 2 min): rt = 1.14 min; m/z = 374.2 [M+H]<+>, peak area >96%.

<1>H nmR (400 MHz, DMSO-d6) δ 11.91 (s, 1H), 10.78 (s, 1H), 9.25 (s, 1H), 8.60 (dd, J = 8.5, 1.2 Hz , 1H), 7.97 (dd, J = 8.0, 1.7 Hz , 1H), 7.64 (ddd, J = 8.7, 7.3, 1.7 Hz , 1H), 7.38 (s, 1H), 7.26 - 7.18 (m, 1H), 6.81 (s, 1H), 4.08 (q, J = 7.1 Hz , 2H), 3.88 (s, 3H), 3.56 (s, 2H), 1.19 (t, J = 7.1 Hz, 3H).

HRMS: [M+H]<+>, calcd. for C19H20NO7: 374.12342, found: 374.12333

Biological data: IIIa-001a-E2: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 33; VEGF-A1 IC50 [µM] = 43; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 69.66; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 44.46

53) (2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid, compound IIIa-001aTz:

UPLC-MS (acid procedure, 2 min): rt = 0.87 min; m/z = 356.1 [M+H], peak area >95%.

<1>H nmR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 11.43 (s, 1H), 10.74 (s, 1H), 9.21 (s, 1H), 8.46 (dd, J = 8.5, 1.2 Hz , 1H), 7.87 (d, J = 8.1 Hz , 1H). 7.60 (td, J = 8.6, 7.9, 1.6 Hz, 1H), 7.39 - 7.30 (m, 2H), 6.80 (s, 1H), 3.48 (s, 2H).

HRMS: [M+H]<+>, calcd. for C16H14N5O5: 356.09894, found: 356.09889

Biological data: IIIa-001aTz: FGF-1 IC50 [µM] = 51; FGF-2 IC50 [µM] = 14; VEGF-A1 IC50 [µM] = 12; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.71; PMN ROS [inhibition at 0.3 µm [%] = 69.35; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 39.74; Whole blood: GM-CSF IC50 [µM] = >100; IFNγ IC50 [µM] = 18.9; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = >100; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 1.98; IL-9 IC50 [µM] = 3.01; IL-10 IC50 [µM] = 7.78; IL-12p70 IC50 [µM] = 0.48; IL-13 IC50 [µM] = 0.15; IL-17A IC50 [µM] = 0.17; IL-17F IC50 [µM] = 0.16; IL-18 IC50 [µM] = 27.7; IL-21 IC50 [µM] = 3; IL-33 IC50 [µM] = 0.14; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.31; TNF β IC50 [µM] = 0.23.

Ethyl ester:

UPLC-MS (acid procedure, 4 min): rt = 1.56 min; m/z = 384.2 [M+H]<+>, peak area >95%.

<1>H nmR (400 MHz, DMSO-d6) δ 11.34 (s, 1H), 10.73 (s, 1H), 9.25 (s, 1H), 8.45 (dd, J = 8.5, 1.2 Hz , 1H), 7.85 (dd, J = 7.8, 1.6 Hz , 1H), 7.61 (ddd, J = 8.7, 7.4, 1.6 Hz , 1H), 7.39 - 7.30 (m, 2H), 6.80 (s, 1H), 4.08 (q, J = 7.1 Hz , 2H), 3.56 (s, 2H), 1.19 (t, J = 7.1 Hz , 3H).

HRMS: [M+H]<+>, calcd. for C18H18N5O5: 384.13024, found: 384.12999

Biological data: IIIa-001aTz-E1: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.32; PMN ROS [inhibition at 0.3 µm [%] = 70.03; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 91.76

54) 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)isophthalic acid, compound IIIa-013a:

UPLC-MS (acid procedure, 4 min): rt = 0.82 min; m/z = 376.0 [M+H]<+>, peak area >89%.

<1>H nmR (400 MHz, DMSO-d6) δ 13.67 - 11.48 (m, 3H), 10.82 (s, 1H), 9.19 (s, 1H), 7.95 (d, J = 7.8 Hz , 2H), 7.36 (s, 1H), 7.33 (t, J = 7.8 Hz , 1H), 6.79 (s, 1H), 3.48 (s, 2H).

HRMS: [M+H]<+>, calcd. for C17H14NO9: 376.06630, found: 376.06638 Biological data: IIIa-013a: FGF-1 IC50 [µM] = 17; FGF-2 IC50 [µM] = 31; VEGF-A1 IC50 [µM] = 43; VEGFR-inhibition of phosphorylation IC50 [µM] =1.3; PMN ROS [inhibition at 0.3 µm [%] = 76.55; PMN ROS inhibition IC50 [µM] = N.D.;

Inhibition of neutrophil adhesion [%] = 30,667; Whole blood: GM-CSF IC50 [µM] = 27.8; IFNγ IC50 [µM] = 0.15; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = 57.1; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 3.55; IL-9 IC50 [µM] = 11.9; IL-10 IC50 [µM] = 37.3; IL-12p70 IC50 [µM] = 0.5; IL-13 IC50 [µM] = 0.37; IL-17A IC50 [µM] = 0.15; IL-17F IC50 [µM] = 0.41; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = 1.63; IL-33 IC50 [µM] = 1.11; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.51; TNF β IC50 [µM] = 0.89.

55) 5-(4-(carboxymethyl)-2,5-dihydroxybenzamido)isophthalic acid, compound IIIa-015a:

UPLC-MS (acid procedure, 4 min): rt = 0.93 min; m/z = 376.0 [M+H]<+>, peak area >96%.

<1>H nmR (400 MHz, DMSO-d6+10% D2O) δ 8.34 (s, 2H), 8.18 (s, 1H), 7.38 (s, 1H), 6.71 (s, 1H), 3.39 (s, 2H).

HRMS: [M+H]<+>, calcd. for C17H14NO9: 376.06630, found: 376.06665

Biological data: IIIa-015a: FGF-1 IC50 [µM] = 16; FGF-2 IC50 [µM] = 114; VEGF-A1 IC50 [µM] = 202; VEGFR-inhibition of phosphorylation IC50 [µM] =0.89; PMN ROS [inhibition at 0.3 µm [%] = 78.76; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 12.13

Ethyl dimethyl ester

UPLC-MS (acid procedure, 4 min): rt = 1.69 min; m/z = 432.1 [M+H]<+>, peak area >97%.

<1>H nmR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.58 (s, 1H), 8.58 (s, 1H), 8.23 - 8.21 (m, 1H), 7.31 (s, 1H), 6.81 (s, 1H), 4.08 (q, J = 7.0 Hz, 2H). 3.91 (s, 6H), 3.58 (s, 2H), 1.19 (t, J = 7.1 Hz , 3H), HRMS: [M+H]<+>, calcd. for C21H22NO9: 432.12891, found: 432.02903

Biological data: IIIa-015a-E3: FGF-1 IC50 [µM] =N.D.; FGF-2 IC50 [µM] = 191; VEGF-A1 IC50 [µM] = 87; VEGFR-inhibition of phosphorylation IC50 [µM] =7.2; PMN ROS [inhibition at 0.3 µm [%] = 91.37; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 61.22

Example 1.9. type IIIb molecules: Diamides from diamines, 1 example

Scheme 15. Synthesis of diamide type IIIb

Procedure: See general procedure from KI-1 steps [3], [4], [5]

For example:

For terms and yields: See Table 3 (Figure 6)

56) 3,5-bis(2,5-dihydroxy-4-carboxymethylbenzoylamino)benzoic acid, compound IIIb-010a:

UPLC-MS (acid procedure, 4 min): rt = 1.08 min; m/z = 539.2.1 [MH]-, peak area 80%.

<1>H nmR (400 MHz, DMSO-d6) δ 10.85 (brs, 2H), 10.57 (brs, 2H), 9.20 (s, 2H), 8.26 (m, 1H), 8.09 (d, J = 2.0 Hz , 2H), 7.36 (s, 2H), 6.82 (s, 2H), 3.50 (s, 4H).

HRMS: [M+H]+, calcd. for C23H17N2O12: 513.07760, found:

513.07774

Triethyl ester

UPLC-MS (acid procedure, 4 min): rt = 1.81 min; m/z = 623.3 [M-H]-, peak area 94%.

<1>H nmR (400 MHz, DMSO-d6) δ 10.87 (s, 2H), 10.65 (s, 2H), 9.23 (s, 2H), 8.31 - 8.28 (m, 1H), 8.10 (d, J = 2.0 Hz , 2H), 7.35 (s, 2H), 6.81 (s, 2H), 4.35 (q, J = 7.1 Hz , 2H), 4.08 (q, J = 7.1 Hz , 4H), 3.58 (s, 4H), 1.35 (t, J = 7.1 Hz , 3H), 1.19 (t, J = 7.1 Hz , 6H).

Biological data: IIIb-010a-E3: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.65; PMN ROS [inhibition at 0.3 µm [%] = ND; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 0

Example 1.10: MOLECULES OF TYPE IIIc. Dimers, 6 examples

Synthetic schemes and procedures

Scheme 16. X=O

General synthetic procedures:

Ether bond formation from KI-10 and KI-11

Ethyl 2,5-dibenzyloxy-4-[(2,5-dibenzyloxy-4-ethoxycarbonylphenyl)methoxymethyl]benzoate [Step 1]: To a solution of HI-11 (340 mg, 0.83 mmol) and KI-10 (250 mg, 0.64 mmol) in DMF (6 mL), cooled in an ice bath, was added portionwise sodium hydride (76 mg, 1.9 mmol). After 1 h, the reaction mixture was poured into saturated aqueous ammonium chloride solution (50 ml) and the material was extracted with EtOAc (3 × 30 ml), the organic phase was further washed with water (2 × 50 ml) and brine (50 ml), dried with sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash column chromatography (Hexane/EtOAc 0 to 20%) to afford the title compound (161 mg, 33% yield) as a brown solid.

Procedures for removing protection:

See General Procedures [4], [5]

For examples

For terms and yields: See Table 4 (removal of protection) (Figure 7)

57) 4-((2,5-dihydroxy-4-carboxyphenyl)methoxymethyl)-2,5-dihydroxybenzoic acid, compound IIIc-060a:

UPLC-MS (acid procedure, 4 min): rt = 0.94 min; m/z = 349.1 [M-H]-, peak area >90%.

<1>H nmR (400 MHz, DMSO-d6) δ 7.20 (s, 2H), 6.92 (s, 2H), 4.56 (s, 4H),

Biological data: IIIc-060a: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 51; VEGF-A1 IC50 [µM] = 114; VEGFR-inhibition of phosphorylation IC50 [µM] =1.3; PMN ROS [inhibition at 0.3 µm [%] = 86.84; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 41.38

General synthetic procedures:

Amine bond formation from KI-12 and KI-13 via KI-10 and KI-11

Ethyl 2,5-bis(benzyloxy)-4-formylbenzoate (KI-12): To a solution of KI-10 (3.4 g, 8.6 mmol) in dichloromethane (50 mL) was added manganese dioxide (4.5 g, 51.9 mmol). The resulting suspension was stirred under reflux for 4 h. The reaction was filtered through a pad of Celite<®>and the solid was washed with dichloromethane (300 ml), the solvent was then removed under reduced pressure to give the title compound (3.2 g, 94% yield) as a yellowish solid.

UPLC-MS (acid procedure, 2 min): rt = 1.33 min; m/z = no ions, peak area >93%

<1>H nmR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 7.56 (s, 1H), 7.54 - 7.26 (m, 11H), 5.28 (s, 2H), 5.20 (s, 2H), 4.30 (q, J = 7.1 Hz , 2H), 1.27 (t, J = 7.1 Hz, 3H).

Ethyl 4-(azidomethyl)-2,5-bis(benzyloxy)benzoate: To a suspension of KI-10 (3.0 g, 7.6 mmol) and 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (1.5 ml, 9.9 mmol) in toluene (30 ml) was added diphenyl phosphoryl azide (DPPA) (2 ml, 9.1 mmol). The resulting mixture was stirred at room temperature for 18 h. Aqueous 1M hydrogen chloride (200 ml) was added and the product was extracted with EtOAc (3 x 100 ml), the combined organic layers were washed with water (2 x 50 ml), dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by flash column chromatography (Hexane/EtOAc 0 to 20%) to afford the title compound (3.1 g, 98%) as a colorless oil which turned white with time.

solid substance.

UPLC-MS (acid procedure, 2 min): rt = 1.37 min; m/z = 390.2, [M+HN2]<+>, peak area >93%

<1>H nmR (400 MHz, DMSO-d6) δ 7.53 - 7.46 (m, 4H), 7.44 - 7.26 (m, 8H), 5.16 (s, 2H), 5.14 (s, 2H), 4.48 (s, 2H), 4.26 (q, J = 7.1, 2H), 1.25 (t, J = 7.1 Hz, 3H).

Ethyl 4-(aminomethyl)-2,5-bis(benzyloxy)benzoate (KI-13): To a solution of ethyl 4-(azidomethyl)-2,5-bis(benzyloxy)benzoate (2.8 g, 6.7 mmol) in THF (60 ml) and water (6 ml) was added polymer-bound triphenylphosphine (4.7 g, ~3 mmol/g charge) and the resulting mixture was allowed to stir at room temperature for 18 h. The resin was removed by filtration and washed with water (100 mL) and EtOAc (2 x 100 mL). The phases were separated and the aqueous layer was further extracted with EtOAc (100 ml), the combined organic layers were washed with brine (200 ml), dried over sodium sulfate and concentrated to give the title compound (KI-13) (1.8 g, 70% yield) as a white solid.

UPLC-MS (basic procedure, 2 min): rt = 1.20 min; m/z = 392.2, [M+H]<+>, peak area >93%

<1>H nmR (400 MHz, DMSO-d6) δ 7.60 - 7.22 (m, 12H), 5.14 (s, 2H), 5.10 (s, 2H), 4.24 (q, J = 7.1 Hz , 2H), 3.75 (s, 2H), 1.25 (t, J= 7.1 Hz , 3H).

Ethyl 2,5-dibenzyloxy-4-[(2,5-dibenzyloxy-4-ethoxycarbonylphenyl)methylaminomethyl] benzoate: KI-12 (500 mg, 1.3 mmol) and KI-13 (641 mg, 1.6 mmol) were dissolved in DCM (35 mL) followed by addition of activated molecular sieves (10 beads). The resulting mixture was stirred at room temperature for 5 h. Sodium triacetoxyborohydride (654 mg, 3.1 mmol) was then added and the resulting mixture was stirred at room temperature for 18 h. DCM (25 mL) was added, the reaction mixture was decanted into a separatory funnel and washed with water (50 mL), dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by flash column chromatography ((Hexane/EtOAc 0 to 50%) to give the title compound (631 mg, 64% yield) as a colorless oil which became a solid over time.

UPLC-MS (acid procedure, 2 min): rt = 1.37 min; m/z = 766.3, [M+H]<+>, peak area >92%

<1>H nmR (400 MHz, DMSO-d6) δ 7.47 - 7.42 (m, 4H), 7.38 - 7.25 (m, 20H), 5.08 (s, 4H), 5.04 (s, 4H), 4.25 (q, J = 7.1 Hz , 4H), 3.76 (s, 4H), 1.25 (t, J = 7.1 Hz, 6H).

Deprotection Procedures: See General Procedures [4], [5]

For terms and yields: See Figure 7.

For examples

58) Bis(4-carboxy-2,5-dihydroxyphenylmethyl)amine, compound IIIc-056a:

OPUPLC-MS (acid procedure, 4 min): rt = 0.57 min; m/z = 350.0 [M+H]<+>, peak area >96%.

<1>H nmR (400 MHz, DMSO-d6) δ 9.97 (s, 2H), 9.08 (s, 2H), 7.31 (s, 2H), 7.02 (s, 2H), 4.11 (s, 4H). HRMS: [M+H]<+>, calcd. for C16H16NO8: 350.08704, found: 350.08706

Biological data: IIIc-056a: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 35; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 90.07; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 35

Scheme 18. X = NAC

General synthetic procedures:

N,N-bis-[2,5-dibenzyloxy-4-ethoxycarbonylphenylmethyl]acetamide: To a solution of N,N-bis-[2,5-dibenzyloxy-4-ethoxycarbonylphenylmethyl]amine (300 mg, 0.39 mmol) and pyridine (0.2 ml, 2.4 mmol) in DCM (6 ml), under a nitrogen atmosphere, was added acetic anhydride (0.2 ml, 2.1 mmol). The reaction mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. The residue was purified by flash column chromatography (Hexane/EtOAc, 0 to 50%) to afford the title compound (316 mg, 92% yield) as a colorless oil.

UPLC-MS (acid procedure, 2 min): rt = 1.44 min; m/z = 808.2 [M+H]<+>, peak area >95%.

<1>H nmR (400 MHz, DMSO-d6) δ 7.49 - 7.17 (m, 22H), 6.92 (s, 1H), 6.72 (s, 1H), 5.03 (s, 2H), 5.00 (s, 2H), 4.96 (s, 2H), 4.94 (s, 2H), 4.46 (s, 4H), 4.31 - 4.16 (m, 4H), 1.96 (s, 3H), 1.28 - 1.20 (m, 6H).

Procedures for removing protection:

See General Procedures [4], [5]

For examples

For terms and yields: See Figure 7 (removal of protection)

59) N,N-Bis(4-carboxy-2,5-dihydroxyphenylmethyl)acetamide, compound IIIc-057a:

UPLC-MS (acid procedure, 4 min): rt = 0.84 min; m/z = 392.1 [M+H]<+>, peak area >99%.

<1>H nmR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 9.34 (s, 1H), 7.22 (s, 1H), 7.17 (s, 1H), 6.63 (s, 1H), 6.53 (s, 1H), 4.48 (s, 2H), 4.39 (s, 2H). 2.11 (s, 3H).

HRMS: [M+H]<+>, calcd. for C18H18NO9: 392.09761, found: 392.09766

Biological data: IIIc-057a: FGF-1 IC50 [µM] = 87; FGF-2 IC50 [µM] = 77; VEGF-A1 IC50 [µM] = 38; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.2; PMN ROS [inhibition at 0.3 µm [%] = 90.34; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 40.04; Whole blood: GM-CSF IC50 [µM] = >100; IFNγ IC50 [µM] = 0.9; IL-1β IC50 [µM] = 16.2; IL-2 IC50 [µM] = 24.2; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.19; IL-9 IC50 [µM] = 11.1; IL-10 IC50 [µM] = >100; IL-12p70 IC50 [µM] = 0.73; IL-13 IC50 [µM] = 4.73; IL-17A IC50 [µM] = 1.32; IL-17F IC50 [µM] = 5.06; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = 1.56; IL-33 IC50 [µM] = 2.93; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.15; TNF β IC50 [µM] = 1.58.

General synthetic procedures:

Tertiary amine formation from KI-11 and NH3

Tris(4-ethoxycarbonyl-2,5-dibenzyloxyphenylmethyl)amine: A sealed tube was charged with KI-11 (32.5 g, 79 mmol), sodium iodide (0.79 g, 5 mmol), ammonia in MeOH (7 N) (38 mL, 264 mmol), and EtOAc (80 mL). The tube was then sealed and the reaction mixture was heated at 60 °C for 24 h. After 24 h, the starting material (KI-11) was consumed and the reaction mixture contained the desired product but also monomeric and dimeric structures. Water (100 mL) was added and the resulting mixture was extracted with EtOAc (3 x 100 mL), the collected organic layer was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The resulting residue was dissolved in EtOAc (165 mL) and KI-11 (2.5 g, 6 mmol), sodium iodide (0.79 g, 5 mmol), DIPEA (10 mL, 58 mmol) were added and the resulting solution was heated to 60 °C. After 18 h, water (100 ml) was added and the resulting mixture was extracted with EtOAc (3 x 100 ml), the collected organic layer was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by recrystallization using EtOH/EtOAc 95/5 (20 mL) as solvent to afford the title compound (15.0 g, 50%) as white crystals.

UPLC-MS (basic procedure, 2 min): rt = 1.71 min; m/z = 1140.3 [M+H]<+>, peak area >99%.

<1>H nmR (400 MHz, DMSO-d6) δ 6.56 - 6.49 (m, 12H), 6.48 - 6.38 (m, 24H), 4.22 (s, 6H), 4.04 (s, 6H), 3.42 (q, J = 7.1 Hz , 6H), 3.00 (s, 6H), 0.42 (t, J = 7.1 Hz, 9H).

Alternatively, the product can be purified by flash chromatography on a silica gel column: scale, up to 270 g; Instrument: Combiflash Torrent; cartridge: RediSep column, silica 3 kg; loading type: Crude solution dissolved in 1 L heptane:toluene (1:1). Elution with ethyl acetate/heptane. Detection: UV - 240 nm

Deprotection Procedures: See General Procedures [4], [5]

For terms and yields: See Figure 7 (removal of protection)

Alternative large-scale deprotection procedures:

Saponification. Tris(4-ethoxycarbonyl-2,5-dibenzyloxyphenylmethyl)amine (95.00 g, 0.083 mol), sodium hydroxide (40.00 g, 0.99 mol), water (570 mL), and tetrahydrofuran (1.9 L) were added to a 5 L round bottom flask. The reaction mixture was heated, using a temperature block, to 80 °C (reflux temperature was 65 °C) for 48 h and then stirred at room temperature for 24 h. The solvents were then removed in vacuo. The crude material was collected and further diluted with water (2.85 L). In another 10 L flask, a mixture of acetic acid (180 ml) and water (950 ml) was stirred at room temperature. A mixture of crude product in water was added to the acetic acid mixture over a period of 30 min. with stirring with the overhead stirrer and a creamy solid precipitated. The reaction mixture was stirred for an additional 1 h and the solid was isolated after filtration; The pH of the mother liquor was 4.2. The isolated solid was stirred with water (1425 mL) for 1 h and then filtered. The pH of the mother liquor was 4.5. The resulting solid was stirred with more water (1425 mL) for 1 h and then filtered; The pH of the mother liquor was 4.5. The above solid was stirred with acetone (950 mL) for 1 h and filtered. The cream-colored substance was dried in a vacuum oven at 50 °C for 48 h before analysis (82.00 g, 93% yield) (yields vary between 90-95%).

Note: pH is a critical parameter during washing to maintain the product in free acid form and remove excess acetic acid from the product.

Ideal pH: 4.2 to 4.5 (final sodium content varies between 2ppm-20ppm).

Hydrogenolysis and isolation. Tris(4-carboxy-2,5-dibenzyloxyphenylmethyl)amine (58.00 g, 54.00 mmol, drug) and tetrahydrofuran (1160 mL) were placed in a 2 L round bottom flask. The mixture was degassed with nitrogen, then washed with a hydrogen/vacuum cycle 4-5 times. Palladium on carbon (JM 10% 424 Pd/C, 70 g, 15% loading) was added to the reaction mixture and stirred at 25 °C for 5-6 h. The reaction was then degassed and the catalyst was removed by filtration through a pad of celite, which was washed with tetrahydrofuran (3×1160 mL). Finally, the filtrates were concentrated under reduced pressure. The crude solid was collected, mixed with heptane (1160 mL) and filtered to give a gray solid, which was dried in a vacuum oven at 50 °C overnight (32.50 g, >100% yield on a crude basis). The collected crude material was dissolved in ethanol (325 ml) and heated to 60 °C (clear solution). Water (325 ml) was then slowly added at 60 °C (maintaining the temperature at least at 50 °C during the addition) over a period of 15-20 min. A cloudy liquid is observed at this stage. The cloudy reaction mixture was stirred at reflux temperature for 30 min.

After this time, the reaction mixture was allowed to cool to room temperature for another 30 min. The precipitated solid was isolated after filtration and washed with 1:1 ethanol:water (66 mL). The solid was dried in a vacuum oven at 50 °C for at least 72 h before analysis to give the desired product IIIc-061a (22.00 g, 76% yield) (yield varies between 65-75%).

LCMS: >95%, nmR: >95% purity.

Tris(4-carboxy-2,5-dihydroxyphenylmethyl)amine, compound IIIc-061a:

UPLC-MS (acid procedure, 4 min): rt = 0.74 min; m/z = 516.0 [M+H]<+>, peak area >86%.

<1>H nmR (400 MHz, DMSO-d6) δ 7.27 (s, 3H), 6.83 (s, 3H), 4.33 (s, 6H).

<13>C nmR (100 MHz, DMSO-d6) δ 172.0, 154.3, 148.3, 134, 117.8 (CH), 114.9 (CH), 112.4, 53.6 (CH2). HRMS: [M+H]<+>, calcd. for C24H22NO12: 516.11365, found: 516.11389

Biological data: IIIc-061a: FGF-1 IC50 [µM] = 9; FGF-2 IC50 [µM] = 7.6; VEGF-A1 IC50 [µM] = 21; VEGFR-inhibition of phosphorylation IC50 [µM] =4.3; PMN ROS [inhibition at 0.3 µm [%] = 92.5; PMN ROS inhibition IC50 [µM] =ND;

Inhibition of neutrophil adhesion [%] = 58.69; Whole blood: GM-CSF IC50 [µM] = 31.2; IFNγ IC50 [µM] = 2.48; IL-1β IC50 [µM] = >100; IL-2 IC50 [µM] = >100; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.18; IL-9 IC50 [µM] = 8.85; IL-10 IC50 [µM] = 17.9; IL-12p70 IC50 [µM] = 37.2; IL-13 IC50 [µM] = 0.31; IL-17A IC50 [µM] = 0.04; IL-17F IC50 [µM] = 0.32; IL-18 IC50 [µM] = 32.4; IL-21 IC50 [µM] = 6.38; IL-33 IC50 [µM] = 0.31; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.03; TNF β IC50 [µM] = 0.81

Triethyl ester

UPLC-MS (acid procedure, 2 min): rt = 1.05 min; m/z = 600.2 [M+H]<+>, peak area >82%.

<1>H nmR (400 MHz, DMSO-d6) δ 10.02 (s, 3H), 9.54 (s, 3H), 7.17 (s, 3H), 7.00 (s, 3H), 4.33 (q, J = 7.1 Hz , 6H), 3.59 (s, 6H), 1.31 (t, J = 7.1 Hz , 9H).

HRMS: [M+H]<+>, calcd. for C30H34NO12: 600.20755, found: 600.20790

Biological data: IIIc-061a-E3: FGF-1 IC50 [µM] = 200; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.34; PMN ROS [inhibition at 0.3 µm [%] = ND; PMN ROS inhibition IC50 [µM] = N.D.; Inhibition of neutrophil adhesion [%] = 10.4

General synthetic procedures:

Formation of a thioether bond from KI-11

See Scheme 21. Same conditions for the formation of the thioether bond.

Deprotection Procedures: See General Procedures [4], [5] For examples

For conditions and yields: See Figure 7 (deprotection) 60) 4-((2,5-dihydroxy-4-carboxyphenyl)methylthiomethyl)-2,5-dihydroxybenzoic acid, compound IIIc-058a:

UPLC-MS (acid procedure, 2 min): rt = 0.84 min; m/z = 365.1 [M-H]-, peak area >79%

<1>H nmR (400 MHz, Methanol-d4) δ 7.25 (s, 2H), 6.83 (s, 2H), 3.69 (s, 4H).

HRMS: [M+H]<+>, calcd. for C16H15O8S: 367.04822, found: 367.04768

Biological data: IIIc-058a: FGF-1 IC50 [µM] = 12; FGF-2 IC50 [µM] = 12; VEGF-A1 IC50 [µM] = 124; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.29; PMN ROS [inhibition at 0.3 µm [%] = 61.22; PMN ROS inhibition IC50 [µM] = 1.17; Inhibition of neutrophil adhesion [%] = 23

General synthetic procedures:

Oxidation of the thioether bond

See preparation IVc-059a: same oxidation conditions.

Deprotection Procedures: See General Procedures [4], [5]

For examples

For terms and yields: See Figure 7 (removal of protection)

61) 4-((2,5-dihydroxy-4-carboxyphenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid, compound IIIc-059a:

UPLC-MS (acid procedure, 2 min): rt = 0.72 min; m/z = 397.1 [M-H]-, peak area >97%

<1>H nmR (400 MHz, DMSO-d6) δ 13.94 (brs, 2H), 10.63 (brs, 2H), 9.71 (s, 2H), 7.27 (s, 2H), 6.91 (s, 2H), 4.44 (s, 4H).

HRMS: [M+H]<+>, calcd. for C16H15O10S: 399.03804, found:

399.03768

Biological data: IIIc-059a: FGF-1 IC50 [µM] = 47; FGF-2 IC50 [µM] = 50; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =3.61; PMN ROS [inhibition at 0.3 µm [%] = 66.39; PMN ROS inhibition IC50 [µM] = 1; Inhibition of neutrophil adhesion [%] = 14.67

Example 1.11: Compounds of type IVc

Scheme 22:

Synthesis via dimethyl ether

General synthetic procedures:

Synthesis from 5-O-methyl gentisic acid

3-formyl-2-hydroxy-5-methoxybenzoic acid.

Hexamethylenetetramine (40.019 g, 0.285 mol) was added to a mixture of 2-hydroxy-5-methoxybenzoic acid (24.0 g, 0.143 mol) in trifluoroacetic acid (190.0 mL). The reaction was refluxed for 18 h. Upon completion, the reaction was cooled to room temperature and a solution of 2M hydrochloric acid (700 ml) was added to the mixture, which was stirred at room temperature for 24 h. The precipitate was filtered, washed with water (500 mL) and dried in a vacuum oven to give 3-formyl-2-hydroxy-5-methoxybenzoic acid as a pale yellow solid (21.30 g, 0.11 mol, 77.0%)

UPLC-MS (acid procedure, 2 min): rt = 0.69 min; m/z = 195.1 [M-H]-, peak area >98%

<1>H nmR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 7.62 (d, J = 3.4 Hz , 1H), 7.44 (d, J = 3.4 Hz , 1H), 3.78 (s, 3H).

Methyl 3-formyl-2,5-dimethoxybenzoate. Methyl iodide (6.7 mL, 0.107 mol) was added to a mixture of 3-formyl-2-hydroxy-5-methoxybenzoic acid (10.0 g, 0.05 mol) and potassium carbonate-325 mesh (28.18 g, 0.20 mol) in DMF (100.0 mL). The resulting mixture was stirred at room temperature for 18 h. The reaction was cooled to room temperature and treated with cold water (400 mL) which resulted in the formation of a precipitate. The resulting suspension was stirred for 10 min, then filtered and dried in a vacuum oven to give methyl 3-formyl-2,5-dimethoxybenzoate as a yellow solid (8.90 g, 0.04 mol, 78.0%).

UPLC-MS (acid procedure, 2 min): rt = 0.95 min; m/z = 225.1 [M+H]<+>, peak area >98%

<1>H nmR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 7.55 (d, J = 3.4 Hz , 1H), 7.41 (d, J = 3.4 Hz , 1H), 3.88 (s, 3H), 3.87 (s, 3H), 3.83 (s, 3H).

Methyl 3-(hydroxymethyl)-2,5-dimethoxybenzoate. Sodium borohydride (16.198 g, 0.428 mol) was slowly added to a solution of methyl 3-formyl-2,5-dimethoxybenzoate (48.0 g, 0.214 mol) in MeOH (900 mL) at 0 °C and the resulting mixture was stirred at ambient temperature for 15 min. The solvent was removed in vacuo and the residue was dissolved in DCM (200 ml) and then washed with water (200 ml). The organic layer was dried over Na2SO4, filtered and concentrated to dryness to give methyl 3-(hydroxymethyl)-2,5-dimethoxybenzoate as a white solid (45.6 g, 0.20 mol, 95.0%)

UPLC-MS (acid procedure, 2 min): rt = 0.84 min; no ionization, peak area >98%

<1>H nmR (400 MHz, DMSO-d6) δ 7.20 (d, J = 3.2 Hz , 1H), 7.08 (d, J = 3.3 Hz , 1H), 5.24 (t, J = 5.7 Hz , 1H), 4.54 (d, J = 5.6 Hz , 2H), 3.83 (s, 3H), 3.76 (s, 3H), 3.68 (s, 3H).

Methyl 3-(chloromethyl)-2,5-dimethoxybenzoate. Thionyl chloride (17.2 mL, 0.235 mol) was added dropwise to a solution of methyl 3-(hydroxymethyl)-2,5-dimethoxybenzoate (45.60 g, 0.20 mol) over 30 min at ambient temperature. The resulting mixture was stirred for 18 h at room temperature. After completion, the reaction mixture was washed with saturated sodium carbonate solution (3 x 500 mL) and brine (500 mL). The organic layer was dried over Na2SO4, filtered and concentrated to dryness to give methyl 3-(chloromethyl)-2,5-dimethoxybenzoate as a brown solid (49.1 g, 0.18 mol, 90%).

UPLC-MS (acid procedure, 2 min): rt = 1.10 min; no ionization, peak area >92%

<1>H nmR (400 MHz, DMSO-d6) δ 7.29 (d, J = 3.2 Hz , 1H), 7.22 (d, J = 3.3 Hz , 1H), 4.74 (s, 2H), 3.85 (s, 3H), 3.77 (s, 3H), 3.76 (s, 3H).

Methyl 3-(acetylthiomethyl)-2,5-dimethoxybenzoate. Potassium thioacetate (15.54 g, 0.136 mol) was added to a solution of methyl 3-(chloromethyl)-2,5-dimethoxybenzoate (22.20 g, 0.091 mol) in THF (500.0 mL) at ambient temperature. The reaction mixture was stirred at 75 °C for 18 h. After completion, the solvent was removed under reduced pressure to give a red oily residue which was treated with brine (500 mL), then extracted with diethyl ether (2 x 250 mL). The organic phases were combined, dried over Na 2 SO 4 , filtered and concentrated to dryness to give methyl 3-(acetylthiomethyl)-2,5-dimethoxybenzoate as a brown solid (25.50 g, 0.09 mol, 99%).

UPLC-MS (acid procedure, 2 min): rt = 1.10 min; no ionization, peak area >92%

<1>H nmR (400 MHz, DMSO-d6) δ 7.12 (d, J = 3.3 Hz , 1H), 7.10 (d, J = 3.2 Hz , 1H), 4.10 (s, 2H), 3.84 (s, 3H), 3.74 (s, 3H), 3.70 (s, 3H), 2.35 (s, 3H).

Methyl 3-(mercaptomethyl)-2,5-dimethoxybenzoate. To a solution of methyl 3-(acetylthiomethyl)-2,5-dimethoxybenzoate (25.5 g, 89.68 mmol) in dry methanol (800 ml) was added dropwise a solution of sodium methanolate (5.81 g, 107.62 mmol) under a nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 45 min before being quenched with Dowex X8(H<+>) ion exchange resin and stirred for 15 min. After filtration, the solvent was evaporated under reduced pressure to give the crude product as a brown oil (containing the desired product and the corresponding disulfide compound in a 2:1 ratio). The residue was dissolved in DMF (400.0 mL) and treated with dithiothreitol (DTT) (33.95 g, 0.26 mol) under nitrogen. The reaction mixture was stirred at 75 °C for 18 h. After the conversion of disulfide to thiol was complete, the solvent was evaporated. The resulting residue was dissolved in DCM (1 L), washed with brine (7 × 500 mL), dried over Na2SO4, filtered and concentrated to dryness to give homogeneous methyl 3-(mercaptomethyl)-2,5-dimethoxybenzoate as a brown oil (21.40 g, 88.32 mmol, 99%).

UPLC-MS (acid procedure, 2 min): rt = 1.05 min; m/z = 243.1 [M+H]<+>, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 7.19 (d, J = 3.2 Hz , 1H), 7.09 (d, J = 3.3 Hz , 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.73 (s, 3H), 3.71 (d, J = 8.0 Hz , 2H), 2.93 (t, J = 8.0 Hz, 1H).

Dimethyl 3,3'-(thiobis(methylene))bis(2,5-dimethoxybenzoate). Triethylamine (19.0 mL, 136.61 mmol) was added to a solution of methyl 3-(mercaptomethyl)-2,5-dimethoxybenzoate (21.40 g, 88.32 mmol) and methyl 3-(chloromethyl)-2,5-dimethoxybenzoate (22.92.74 mmol) under a nitrogen atmosphere. The reaction was stirred at room temperature for 18 h. After completion, the solvent was removed under vacuum to give a brown oil residue which was treated with water (1 L), then extracted with diethyl ether (3 × 500 mL). The organic phases were combined, washed with brine (5 x 250 ml), dried over Na 2 SO 4 , filtered and concentrated to dryness to give the crude product as a brown oil, which was purified by chromatography on silica gel: elution was made with a gradient of ethyl acetate (0 to 20%) in isohexane to give dimethyl 3,3'-(thiobis(methylene))bis(2,5-dimethoxybenzoate) as a brown solid (29.9 g, 66.37 mmol, 76%)

UPLC-MS (acid procedure, 2 min): rt = 1.22 min; m/z = 451.2 [M+H]<+>, peak area >92%

<1>H nmR (400 MHz, DMSO-d6) δ 7.12 - 7.08 (m, 4H), 3.83 (s, 6H), 3.76 (s, 4H), 3.73 (s, 6H), 3.67 (s, 6H).

3,3'-(thiobis(methylene))bis(2,5-dimethoxybenzoic acid). A solution of lithium hydroxide monohydrate (1.0 g, 23.83 mmol) in water (30.0 mL) was added to a solution of dimethyl 3,3'-(thiobis(methylene))bis(2,5-dimethoxybenzoate) (2.0 g, 4.44 mmol) in THF (100.0 mL). The reaction was stirred at room temperature for 48 h. After completion, water (100 ml) was added to the reaction and the mixture was washed with ethyl acetate (100 ml).

The aqueous layer was acidified with 1 M aqueous hydrochloric acid to pH=2 and extracted with ethyl acetate (3 x 100 ml). The organic layers were collected, dried (Na2SO4), filtered and concentrated to give 3,3'-(thiobis(methylene))bis(2,5-dimethoxybenzoic acid) as a brown solid (1.91 g, 4.11 mmol, 93%)

UPLC-MS (acid procedure, 2 min): rt = 0.93 min; m/z = 421.1 [M-H]-, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 12.96 (s, 2H), 7.10 (d, J = 3.3 Hz , 2H), 7.08 (d, J = 3.2 Hz , 2H), 3.76 (s, 4H), 3.73 (s, 6H), 3.69 (s, 6H).

3-[(2,5-Dihydroxy-3-carboxyphenyl)methylthiomethyl]-2,5-dihydroxybenzoic acid. To a solution of the previous thioether (5.0 g, 0.01 mol) in DCM (200.0 mL) was slowly added a 1M solution of tribromoborane in DCM (100.7 mL, 0.101 mol) at 0 °C and the reaction mixture was refluxed for 48 h. After this time, the solid was filtered, washed with DCM (500 ml) and then suspended in 1M hydrochloric acid solution (50 ml). The resulting mixture was refluxed for 2 h. The resulting brown suspension was filtered and dried to give the crude product, which was purified by reverse phase column chromatography (25 g, MeCN:H2O 5% to 95% at 12 CV) to give 2,5-dihydroxy-3-[(2,5-dihydroxy-3-carboxyphenyl)methylthiomethyl]benzoic acid as a white solid (1.1 g, 0.003 mol, 26%).

UPLC-MS (acid procedure, 2 min): rt = 0.70 min; m/z = 365.1 [M-H]-, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 13.86 (s, 2H), 11.09 (s, 2H), 9.14 (s, 2H), 7.08 (d, J = 3.1 Hz , 2H), 7.02 (d, J = 3.1 Hz , 2H), 3.65 (s, 4H).

Example:

62) 3-((2,5-dihydroxy-3-carboxyphenyl)methylthiomethyl)-2,5-dihydroxybenzoic acid, compound IVc-058a:

UPLC-MS (acid procedure, 2 min): rt = 0.70 min; m/z = 365.1 [M-H]-, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 13.86 (s, 2H), 11.09 (s, 2H), 9.14 (s, 2H), 7.08 (d, J = 3.1 Hz , 2H), 7.02 (d, J = 3.1 Hz , 2H), 3.65 (s, 4H).

HRMS: [M+H]<+>, calcd. for C16H15O8S: 367.04822, found: 367.04734 Biological data: IVc-058a: FGF-1 IC50 [µM] = 36; FGF-2 IC50 [µM] = 4.9; VEGF-A1 IC50 [µM] = 83; VEGFR-inhibition of phosphorylation IC50 [µM] = 0.53; PMN ROS [inhibition at 0.3 µm [%] = 77.04; PMN ROS inhibition IC50 [µM] = 0.29; Inhibition of neutrophil adhesion [%] = 43.5; Whole blood: GM-CSF IC50 [µM] = >100; IFNγ IC50 [µM] = 10.6; IL-1β IC50 [µM] = 27.5; IL-2 IC50 [µM] = 3.82; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.21; IL-9 IC50 [µM] = 31.1; IL-10 IC50 [µM] = 16.4; IL-12p70 IC50 [µM] = 4.23; IL-13 IC50 [µM] = 55.9; IL-17A IC50 [µM] = 0.1; IL-17F IC50 [µM] = 86.8; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = 3.2; IL-33 IC50 [µM] = 33.9; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.18; TNF β IC50 [µM] = 59.3.

Synthesis from compound IVc-058a

3-((2,5-Dihydroxy-3-carboxyphenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid. To a solution of thioether IVc-058a (4.25 g, 9.28 mmol) in DMF (20.0 mL) was slowly added a solution of 3-chlorobenzene-1-carboperoxy acid (mCPBA degree of purity ≤77%, 5.80 g, 25.88 mmol) in DMF (20.0 mL). The reaction mixture was stirred for 18 h at room temperature. The crude product was subjected to HPLC purification (see general conditions) to give a white solid (3.20 g) which was triturated with 1M HCl (50 ml) to give the desired product as a white solid (2.55 g, 6.40 mmol, 56%)

UPLC-MS (acid procedure, 4 min): rt = 0.68 min; m/z = 397.1 [M-H]-, peak area >95%

63) 3-((2,5-dihydroxy-3-carboxyphenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid, compound IVc-059a:

UPLC-MS (acid procedure, 4 min): rt = 0.68 min; m/z = 397.1 [M-H]-, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 11.24 (s, 1H), 9.32 (s, 1H), 7.21 (d, J = 3.1 Hz , 1H), 7.09 (d, J = 3.1 Hz , 1H), 4.44 (s, 2H).

<13>C nmR (100 MHz, DMSO-d6) δ 172.4, 153.4, 149.2, 117.4, 116.1 (CH), 113.3 (CH), 53.6 (CH2). HRMS: [M+H]<+>, calcd. for C16H15O10S: 399.03804, found: 399.03775

Biological data: IVc-059a: FGF-1 IC50 [µM] = 6.7; FGF-2 IC50 [µM] = 2.7; VEGF-A1 IC50 [µM] = 12; VEGFR-inhibition of phosphorylation IC50 [µM] =4.8; PMN ROS [inhibition at 0.3 µm [%] = 74; PMN ROS inhibition IC50 [µM] = 0.147;

Inhibition of neutrophil adhesion [%] = 10; Whole blood: GM-CSF IC50 [µM] = >100; IFNγ IC50 [µM] = 4.34; IL-1β IC50 [µM] = 38.6; IL-2 IC50 [µM] = 21.2; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.11; IL-9 IC50 [µM] = 0.4; IL-10 IC50 [µM] = 1.31; IL-12p70 IC50 [µM] = 42; IL-13 IC50 [µM] = >100; IL-17A IC50 [µM] = 0.16; IL-17F IC50 [µM] = >100; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = 1.87; IL-33 IC50 [µM] = 27.5; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.16; TNF β IC50 [µM] = 0.33

Dimethyl ester

UPLC-MS (acid procedure, 2 min): rt = 0.94 min; m/z = 427.1 [M+H]<+>, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 10.28 (s, 2H), 9.43 (s, 2H), 7.21 (d, J = 3.1 Hz , 2H), 7.13 (d, J = 3.1 Hz , 2H), 4.47 (s, 4H), 3.91 (s, 6H).

HRMS: [M+H]<+>, calcd. for C18H19O10S: 427.06934, found: 427.06942

Biological data: IVc-059a-E2: FGF-1 IC50 [µM] = 78; FGF-2 IC50 [µM] = 94; VEGF-A1 IC50 [µM] = 200; VEGFR-inhibition of phosphorylation IC50 [µM] =2.7; PMN ROS [inhibition at 0.3 µm [%] = 45.67; PMN ROS inhibition IC50 [µM] = 2.64; Inhibition of neutrophil adhesion [%] = 50.5

Dimethyl ester tetraacetate

UPLC-MS (acid procedure, 2 min): rt = 1.05 min; m/z = 593.1 [M-H]-, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 7.75 (d, J = 2.8 Hz , 2H), 7.53 (d, J = 2.9 Hz , 2H), 4.65 (s, 4H), 3.81 (s, 6H), 2.31 (s, 6H), 2.23 (s, 6H).

HRMS: [M+H]<+>, calcd. for C26H27O14S: 595.11160, found: 595.11149

Biological data: IVc-059a-E2-A4: FGF-1 IC50 [µM] = 54; FGF-2 IC50 [µM] = 200; VEGF-A1 IC50 [µM] = 25; VEGFR-inhibition of phosphorylation IC50 [µM] =ND; PMN ROS [inhibition at 0.3 µm [%] = 29.17; PMN ROS inhibition IC50 [µM] = 4.13; Inhibition of neutrophil adhesion [%] = 87.5; Whole blood: GM-CSF IC50 [µM] = 1.83; IFNγ IC50 [µM] = 3.5; IL-1β IC50 [µM] = 14.1; IL-2 IC50 [µM] = 10.4; IL-4 IC50 [µM] = >100; IL-5 IC50 [µM] = >100; IL-6 IC50 [µM] = 0.99; IL-9 IC50 [µM] = 2.9; IL-10 IC50 [µM] = 16.8; IL-12p70 IC50 [µM] = 51.4; IL-13 IC50 [µM] = 18.7; IL-17A IC50 [µM] = 0.02; IL-17F IC50 [µM] = 25.8; IL-18 IC50 [µM] = >100; IL-21 IC50 [µM] = >100; IL-33 IC50 [µM] = 23.2; TGFβ IC50 [µM] = >100; TNFα IC50 [µM] = 0.3; TNF β IC50 [µM] = 19.4

An alternative, scalable synthesis of IVc-059a

Scheme 24: Synthesis of IVc-059a from 2,5-dibenzyloxyphthalaldehyde

2,5-bis(benzyloxy)-3-(hydroxymethyl)benzaldehyde [Step 1]: 2,5-bis(benzyloxy)-isophthalaldehyde (25.0 g, 72.1 mmol) (see Scheme 9b) was dissolved in THF (150 mL) and EtOH (15 mL) was added. The brown solution was kept under a positive nitrogen flow and stirred at room temperature for 10 min, then solid NaBH4 (695 mg, 18.37 mmol) was added in portions over 20 min, the internal temperature of the reaction was not allowed to exceed 25 °C (a water bath was used).

The reaction mixture was allowed to stir at room temperature for 30 min, then an additional amount of NaBH 4 was added every 15 min until complete conversion of the starting material (total: 107 mg, in two portions). The reaction mixture was then poured into water (1.2 L) and stirred for 20 min. The brown precipitate thus formed was isolated by filtration and the resulting solid was further dried in a vacuum oven at 40 °C to constant weight to give the title compound (22.9 g, 76% corrected yield) as a brown oil. The nmR profile showed 83% desired product, 15% over-reduced diol, 2% starting material, the material was carried to the next step without further purification.

UPLC-MS (acidic procedure, 2 min): rt = 1.19 min, 86%, m/z = 347.2 [MH]-

1H nmR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 7.52 - 7.28 (m, 11H), 7.19 (d, J = 3.3 Hz , 1H), 5.34 (t, J = 5.6 Hz , 1H), 5.15 (s, 2H) 4.99 (s, 2H), 4.60 (d, J = 5.6 Hz, 2H).

2,5-Bis(benzyloxy)-3-(hydroxymethyl)benzoic acid [Step 2]: 2,5-Bis(benzyloxy)-3-(hydroxymethyl)benzaldehyde (44.3 g, 103 mmol, 81% purity) and KH2PO4 (25.9 g, 190 mmol) were dissolved in acetone (440 mL) and water (130 mL).

Resorcinol (21.0 g, 191 mmol) was added and the reaction mixture was stirred at room temperature for 10 min. A solution of sodium chlorite (21.5 g, 191 mmol) in water (60 mL) was added slowly over 30 min, not allowing the internal temperature to exceed 25 °C. The reaction mixture was stirred at room temperature for 3 h and 2 M aqueous H 3 PO 4 (95 ml) was added. The mixture was stirred for 20 min and cooled to 6 °C in an ice bath before being filtered. The solid was further washed with water until the pH of the filtrate was ~6. The obtained solid was suspended in water (ca. 300 ml) and a pre-cooled (5 - 10 °C) solution of NaOH (17 g, 412 mmol) in water (200 ml) was added. The suspension was stirred at room temperature for 30 min before being filtered onto a sintering funnel. The isolated solid was further washed with 1 M aqueous NaOH (2 x 50 ml) and water (50 ml).

The alkaline liquor was then acidified using 6N aqueous HCl pre-cooled (5 - 10 °C) to pH ~1. The resulting suspension was magnetically stirred for 20 min before filtration, the resulting solid was further dried in a vacuum oven at 40 °C to constant weight to afford the title compound as a light beige solid (34.8 g, 88%).

UPLC-MS (acidic procedure, 2 min): rt = 1.06 min, 99%, m/z = 363.2 [MH]-.

1H nmR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 7.51 - 7.31 (m, 10H), 7.28 (d, J = 3.2 Hz , 1H), 7.22 (d, J = 3.3 Hz , 1H), 5.21 (s, 1H), 5.12 (s, 2H), 4.88 (s, 2H), 4.53 (s, 2H).

Ethyl 2,5-bis(benzyloxy)-3-(hydroxymethyl)benzoate [Step 3]: 2,5-Bis(benzyloxy)-3-(hydroxymethyl)benzoic acid (32.5 g, 89.3 mmol) and Cs2CO3 (32.3 g, 99.2 mmol) were suspended in DMF (200 mL) and ethyl iodide (9.0 mL, 112 mmol) was added to the reaction mixture. mixture. After stirring at room temperature for 3 h, the reaction mixture was poured into saturated aqueous NH 4 Cl (1 L) and solid NaCl (~30 g) was added. After stirring for 10 min, the material was extracted with EtOAc (2 x 500 mL). The collected organic layer was washed with brine (2 x 500 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a brown oil residue (42.0 g, crude, 89.3 mmol). The crude material was carried on to the next step without further purification.

UPLC-MS (acid procedure, 2 min): rt = 1.24 min, 93%, m/z = weak ionization.

1H nmR (400 MHz, DMSO-d6) δ 7.51 - 7.32 (m, 10H), 7.31 (d, J = 3.3 Hz , 1H), 7.22 (d, J = 3.3 Hz , 1H), 5.24 (t, J = 5.6 Hz , 1H), 5.13 (s, 2H), 4.87 (s, 2H), 4.54 (d, J = 5.6 Hz , 2H), 4.26 (q, J = 7.1 Hz , 2H), 1.24 (t, J = 7.1 Hz , 3H)

Ethyl 2,5-bis(benzyloxy)-3-(chloromethyl)benzoate (HI-14) [Step 4]: Ethyl 2,5-bis(benzyloxy)-3-(hydroxymethyl)benzoate (42 g crude material from previous step, 89.3 mmol) was dissolved in DCM (200 mL) under inert atmosphere (N2), then SOCl2 (9.8 mL, 133.9 mmol) was added at 0 °C. (ice bath) and the reaction mixture was allowed to warm to room temperature. After 2 h the volatiles were removed under reduced pressure followed by azeotropic distillation using toluene (3 × 100 mL) to give a semi-solid brown oil (38.0 g, crude, 89.3 mmol).

UPLC-MS (acid procedure, 2 min): rt = 1.40 min, 91%, m/z = weak ionization.

1H nmR (400 MHz, DMSO-d6) δ 7.54 - 7.26 (m, 12H), 5.14 (s, 2H), 4.95 (s, 2H), 4.71 (s, 2H), 4.28 (q, J = 7.1 Hz , 2H), 1.28 - 1.22 (t, J = 7.1 Hz, 3H).

Bis(2,5-dibenzyloxy-3-ethoxycarbonylphenylmethyl)sulfide [step 5]: To a solution of KI-14 (38.0 g, crude material, 89.3 mmol) in DMF (420 mL) was added thioacetamide (8.4 g, 111.8 mmol) followed by the addition of K2CO3 - 325 Mesh (16.7 g, 120.8 mmol). The reaction was heated at 45 °C for 48 h. The reaction was cooled to room temperature, followed by the addition of thioacetamide (2.5 g, 33.3 mmol) and K 2 CO 3 - 325 mesh (5.0 g, 36.1 mmol) and further stirring at 45 °C for 18 h to complete the reaction. The reaction mixture was poured into water (4 l). The reaction mixture was stirred for 20 min before adding EtOAc (2 L) followed by solid NaCl (~60 g). The phases were separated followed by additional extraction using EtOAc (1.5 L). The collected organic layer was washed with brine (1.5 L), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the crude title compound (39.8 g, 44.6 mmol) as a brown oil which was used in the next step without purification.

UPLCMS (acid procedure, 2 min): rt = 1.57 min, m/z = 800.5 [M+NH4]+, peak area 77%.

1H nmR (400 MHz, DMSO-d6) δ 7.46 - 7.17 (m, 24H), 5.05 (s, 4H), 4.83 (s, 4H), 4.23 (q, J = 7.1 Hz , 4H), 3.73 (s, 4H), 1.20 (t, J = 7.1 Hz , 6H).

Bis(2,5-dibenzyloxy-3-ethoxycarbonylphenylmethyl)sulfone [Step 6]: To a solution of bis(2,5-dibenzyloxy-3-ethoxycarbonylphenylmethyl)sulfide (39.8 g crude, 44.6 mmol) in acetic acid (850 mL) was slowly added 30 wt% hydrogen peroxide solution (50 mL, 490 mmol). The reaction was stirred at room temperature for 18 h.

The reaction mixture was poured into ice/water (5 L) and stirred at room temperature for 30 min, then EtOAc (2 L) and solid NaCl (~40 g) were added. After phase separation, the aqueous layer was extracted once more with EtOAc (1 L). The collected organic layer was washed with brine (1.5 L), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a brown solid. The solid was suspended in MTBE (250 ml) and magnetically stirred for 30 min, followed by filtration and further trituration with MTBE (150 ml) to give the title compound (23.56 g, 28.9 mmol, 64% yield in step 4).

UPLC-MS (acid procedure, 2 min): rt = 1.48 min; m/z = 832.5 [M+NH4]+, peak area 96%

1H nmR (400 MHz, DMSO-d6) δ 7.50 - 7.24 (m, 24H), 5.08 (s, 4H), 4.85 (s, 4H), 4.50 (s, 4H), 4.26 (q, J = 7.1 Hz , 4H), 1.25 - 1.17 (m, 6H).

Bis(2,5-dibenzyloxy-3-carboxyphenylmethyl)sulfone [Step 7]: A solution of lithium hydroxide (9.7 g, 233 mmol) in water (100 mL) was added to a solution of bis(2,5-dibenzyloxy-3-ethoxycarbonylphenylmethyl)sulfone (33.3 g, 38.8 mmol) in THF (350 mL) and the resulting mixture was stirred at reflux for 18 h. After this time, most of the THF was removed under reduced pressure resulting in a white suspension. After addition of water (3 L) and aqueous sodium hydroxide solution (1 M, 1 L) the mixture remains a suspension. The suspension was stirred at room temperature for 18 h and the aqueous layer was acidified to pH-1 by adding 6 M aqueous hydrochloric acid and the resulting solid was collected by filtration and washed with water, until the liquids were neutral, the resulting white solid was further dried in a vacuum oven at 40 °C to constant weight to give the title compound as a white solid (29.4 g, 38.7 mmol, 99%).

UPLC-MS (acid procedure, 2 min): rt = 1.39 min; m/z = 757.1 [MH]-, peak area 100%

1H nmR (400 MHz, DMSO-d6) δ 13.23 (s, 2H), 7.47 - 7.25 (m, 24H), 5.08 (s, 4H), 4.88 (s, 4H), 4.48 (s, 4H).

Bis(2,5-dihydroxy-3-carboxyphenylmethyl)sulfone [Step 8], IVc-059a: To a suspension of bis(2,5-dibenzyloxy-3-carboxyphenylmethyl)sulfone (10.08 g, 13.28 mmol) in EtOH (140 mL) and THF (140 mL) was added palladium on charcoal (20 wt%, 2 g) as a suspension in water. ml). After several cycles of vacuum/hydrogen, the mixture was maintained in an H2 atmosphere at 25 °C and stirred for 20 h. After completion, the mixture was filtered through Celite using THF. The filtrate was concentrated under reduced pressure to give an off-white solid. The residue was then suspended in acetonitrile (100 ml) and heated to reflux with magnetic stirring, the mixture was then cooled to -5 °C before being filtered and further washed with cold acetonitrile. The product was further dried in a vacuum oven at 40 °C to constant weight to give the desired product as a white solid (5.35 g, 12.78 mmol, 96%).

UPLC-MS (acid procedure, 4 min): rt = 0.67 min; m/z = 397.1 [MH]-, peak area 100%

NMR data: see above.

Example 1.12 - Type V molecules: Reference compounds

Diamides derived from 5-hydroxyisophthalic acid

2 examples

Synthetic schemes and procedures

Scheme 25: synthesis of V-001a

Amide bond formation

See General Procedure B for the coupling of amide [3B] using aniline A (2 moles) or methyl 5-aminosalicylate

Deprotection Procedures: See General Procedures [4], [5]

See Figure 8 for details

For examples

64) bis-N-(4-carboxy-2,5-dihydroxyphenyl)amide of 5-hydroxyisophthalic acid (V-001a)

UPLC-MS (acid procedure, 2 min): rt = 0.72 min; m/z = 483.0 [M+H]<+>, peak area >95%

<1>H nmR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 9.83 (s, 2H), 9.48 (s, 2H), 7.92 (s, 1H), 7.68 (s, 2H), 7.52 (d, J = 1.5 Hz , 2H), 7.30 (s, 2H).

Diethyl ester

UPLC-MS (acid procedure, 2 min): rt = 1.09 min; m/z = 539.2 [M-H]-, peak area 94%

<1>H nmR (400 MHz, DMSO-d6) δ 10.23 (s, 2H), 9.92 (s, 2H), 9.49 (s, 2H), 7.91 (s, 1H), 7.73 (s, 2H), 7.51 (d, J = 1.5 Hz , 2H), 7.32 (s, 2H), 4.35 (q, J = 7.1 Hz , 4H), 1.34 (t, J = 7.1 Hz , 6H).

65) bis-N-(3-carboxy-4-hydroxyphenyl)amide of 5-hydroxyisophthalic acid (V-002a)

UPLC-MS (acid procedure, 4 min): rt = 1.00 min; m/z = 451.1 [M-H]-, peak area 97%

<1>H nmR (400 MHz, DMSO-d6) δ 10.31 (s, 2H), 10.11 (s, 1H), 8.28 (d, J = 2.7 Hz , 2H), 7.98 (m, 1H), 7.89 (dd, J = 9.0, 2.7 Hz , 2H), 7.50 (d, J = 1.5 Hz , 2H), 6.97 (d, J = 9.0 Hz , 2H).

Gentisic acid units linked to urea 1 example

Synthetic scheme and procedures

Scheme 26: synthesis of V-003a

Formation of urea bond

Procedure from KI-1:

N,N'-Bis(2,5-dibenzyloxy-4-ethoxycarbonylphenyl)urea. To a cooled solution (ice bath) of 2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoic acid (HI-1) (2.0 g, 4.9 mmol) and Et3N (1.6 mL, 11.3 mmol) in THF (25 mL) under an inert atmosphere (N2), was slowly added DPPA (1.1 mL, 5.2 mmol). The mixture was slowly warmed to room temperature and stirred at this temperature for 3 h. Then tBuOH (8 mL) was added and the reaction mixture was stirred at room temperature for 18 h. The reaction profile showed most of the urea coupling products instead of the desired Boc-aniline. The mixture was poured into saturated sodium hydrogen carbonate solution (250 mL) and stirred for 5 min before being extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give a colorless oil. The residue was purified by flash column chromatography (iso-hexane/EtOAc 1:0 followed by a gradient to 30% EtOAc) to give the title compound (1.1 g, 56%) as an off-white solid.

UPLC-MS (basic procedure, 2 min): rt = 1.50 min; m/z = 781.2 [M+H]<+>, peak area 85%

<1>H nmR (400 MHz, DMSO-d6) δ 9.49 (s, 2H), 8.18 (s, 2H), 7.58 - 7.45 (m, 8H), 7.43 - 7.35 (m, 8H), 7.35 - 7.28 (m, 6H), 5.27 (s, 4H), 5.11 (s, 4H), 4.22 (q, J = 7.1 Hz , 4H), 1.24 (t, J = 7.1 Hz , 6H).

Deprotection Procedures: See General Procedures [4], [5]

See Figure 8 for details

For example

66) N,N'-Bis(2,5-dihydroxy-4-carboxyphenyl)urea (V-003a)

UPLC-MS (acid procedure, 4 min): rt = 0.90 min; m/z = 363.1 [M-H]-, peak area 93%

<1>H nmR (400 MHz, DMSO-d6) δ 13.36 (s, 2H), 10.86 (s, 2H), 9.64 (s, 2H), 9.48 (s, 2H), 7.77 (s, 2H), 7.17 (s, 2H).

Diethyl ester

UPLC-MS (acid procedure, 4 min): rt = 1.79 min; m/z = 419.2 [M-H]-, peak area 97%

<1>H nmR (400 MHz, DMSO-d6) δ 10.26 (s, 2H), 9.74 (s, 2H), 9.53 (s, 2H), 7.81 (s, 2H), 7.20 (s, 2H), 4.32 (q, J = 7.1 Hz), 1.33 (t, J = 7.1 Hz, 4H). 6H).

Example 1.13: Salt formation

a) Substances.

General protocol: salts were obtained as solids by adding the appropriate number of equivalents of base to solutions or suspensions of the desired compounds in water, sonicating until complete dissolution, adjusting the pH as needed to values between 7.0 and 7.4 by adding aqueous HCl or excess base, then lyophilizing the solution. Ateratively determined salts can be obtained by dissolving in DMSO followed by precipitation with EtOH (IIc-007a). The substances are obtained from the following bases: sodium hydroxide, diethylamine (DEA), lysine (Lis), meglumine (Meg), triethanolamine (TEA).

For examples:

Sodium and diethylammonium (DEA) salts Ic-007a, IIc-007a, IIIc-061a as solids were obtained according to the general protocol without pH adjustment.

- Approximate solubilities in water:

Ic-007a-DEA2: < 10 mg/ml

Ic-007a-DEA3: > 20 mg/ml

IIc-007a-DFA1: > 10 mg/ml

IIc-007a-DEA2: > 20 mg/ml

IIIc-061a-DEA3: > 25 mg/ml

- Stability of free acids and salts as substances:

The free acids Ic-007a, IIc-007a, IIIc-061a and the following salts Ic-007a-DEA2, IIc-007a-DEA2, IIIc-061a-DEA3 were stable as a solid for up to 56d at temperatures of 4 °C, 20 °C and 40 °C; salt Ic-007a-DEA3 was stable at the same temperatures for up to 36 d.

b) Solutions

General protocol: salts were obtained as solutions by adding the appropriate number of equivalents of base to solutions or suspensions of the desired compounds in water, sonicating until complete dissolution, adjusting the pH as needed to values between 7.0 and 7.4 by adding aqueous HCl or excess base. The following bases were tested: ethylamine, triethylamine, ethylenediamine, diethanolamine, triethanolamine, choline, meglumine, lysine and arginine.

For examples:

Salts Ic-007a:

Soluble salts were obtained with meglumine, lysine and diethanolamine after stirring the mixture for 2 days at 40 °C. The solid form of the Ic-007a-Lis2 salt was obtained from DMSO by ethanol precipitation.

The salt was stable as a solution in water for at least 7 days at room temperature.

Salts IIc-007a:

DEAL-salt: on a small scale, the mono-diethylammonium salt was obtained by a general procedure without pH adjustment. Solubility: 17.4 mg/ml vs. 1.8 mg/ml for native diacid

Large scale process and characterization:

IIc-007a (20 g, 42.7 mmol) was charged with EtOH (200 mL). The reaction was stirred at RT to obtain a suspension. DEA (4.4 ml, 42.7 mmol) was charged and the reaction was heated at 50 °C for 2 h. The batch was cooled to room temperature and the solid was filtered. The filter cake was washed with EtOH (50 mL) and dried in an oven at 40 °C to give 21.5 g of the mono-DEA salt as a yellow solid in 98% yield. nmR: 1.6% EtOH, 1.04 eq. DEA. HPLC: 99.6%.

Solid state data for mono-DEA salt IIc-007a showed a clear crystal pattern in XRPD analysis. The TGA trace showed the loss of a small amount of EtOH <120 °C indicating that EtOH can be dried at 60-80 °C. A loss of DEA (theory 13.5 wt% for 1:1 salt) was then observed associated with a melting endotherm in DSC (peak at 273.8 °C), followed by melting of the free acid form. DEA2-salt, pH adjusted: obtained by general procedure with pH adjustment (0.51 additional eq. Et2NH needed to reach pH=7.25). The salt in solution shows clear degradation after 21 days at 40 °C and extensive degradation after 24 h at 80 °C.

DEA2-salt, pH not adjusted: obtained by general procedure without pH adjustment. pH of the solution = 5.87. The salt in solution showed no degradation after 21 days at 4 °C, 20 °C and 40 °C.

Lys2-sol, pH not adjusted: obtained by general procedure using 2 eq. L-lysine. pH of the solution = 5.74.

NOTE: by analogy with the bis (N-methyl)amide of 2-hydroxyisophthalic acid (pKa of phenol = 6.81), the 2-OH group of the isophthalic bis-amide of the core IIc-007a is extremely acidic and its ionization leads to the degradation of the molecule. This effect was not observed for Ic-007a (expected pKa of phenolic functions in the range of 9.8-10.0). Therefore, it is important not to adjust the pH of the IIc-007a didication salt solution.

Salts IIIc-061a:

DEA3-salt, pH adjusted: obtained by general procedure using 3 eq. Et2NH with pH adjustment (0.8 additional eq. HCl (0.5 M) required to reach final pH=7.22). The salt in solution showed no degradation after 21 days at 4 °C, 20 °C and slight degradation at 40 °C after 33 days.

Meg3-salt, pH adjusted: obtained by general procedure using 3 eq. meglumine with pH adjustment to achieve final pH=7.22 (HCl 0.5M). The salt in solution showed no degradation after 21 days at 4 °C, 20 °C and 40 °C after 14 days.

Salts IVc-059a,

DEA2-salt, pH adjusted: obtained by general procedure with pH adjustment (0.32 additional eq. Et2NH needed to reach pH=7.3). The salt in solution shows stability after 26 days at 4 °C, 20 °C and 40 °C.

Example 1.14: Prodrugs

Prodrugs Ic-007a

1) 2-Morpholinoethyl 5-[[2,5-dihydroxy-4-[[4-hydroxy-3-(2-morpholinoethoxycarbonyl) phenyl]carbamoyl]benzoyl]amino]-2-hydroxy-benzoate (Ic-007a-mpe2)

Esterification. A suspension of 5-[[2,5-dibenzyloxy-4-[(3-carboxy-4-hydroxyphenyl)carbamoyl]-benzoyl]amino]-2-hydroxy-benzoic acid (130 mg, 0.2 mmol) in N,N -dimethylformamide (4 ml) was stirred at room temperature. N,N-Dimethylpyridin-4-amine (49 mg, 0.4 mmol) and 2-morpholinoethanol solution were added.

(53 mg, 0.4 mmol) in N,N-dimethylformamide (0.5 mL). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (115 mg, 0.6 mmol) was added and the reaction mixture was heated in a microwave reactor at 60 °C for 1.5 h. The reaction mixture was filtered, the filtrate was diluted with tetrahydrofuran and the solvent was evaporated. The residue was purified by silica gel chromatography eluting with 0-40% methanol in dichloromethane to afford the title product as a white solid (80 mg, 45%). LCMS (m/z) [M+H] 875.1.

Hydrogenolysis: A solution of the above diester (80 mg, 0.092 mmol) in tetrahydrofuran (12 ml) and water (4 ml) was prepared and 10% palladium on carbon (50 mg, 10% paste) was added. The reaction mixture was stirred under a hydrogen atmosphere for 17 hours. The catalyst was removed by filtration and the solvent was evaporated. The residue was purified by reverse phase preparative HPLC on a C18 column using a gradient of 10-97% (acetonitrile 0.1% formic acid): (water 0.1% formic acid).

The residue was triturated with diethyl ether and dried under vacuum to give the title product Ic-007a-mpe2 as a yellow solid (22 mg, 35%).

<1>H nmR (d6-DMSO ppm) δ 11.16-11.00 (br s, 1H), 10.46 (s, 1H), 10.45-10.15 (br s, 1H), 8.25 (d, J=2.7 Hz , 1H), 7.77 (dd, J=9.0, 2.7Hz, 1H), 7.53 (s, 1H), 7.01 (d, J=9.0 Hz , 1H), 4.46 (t, J=5.4 Hz , 2H), 3.62-3.56 (m, 4H), 2.77-2.69 (m, 2H), 2.56-2.50 (m, 4H, partially obscured by DMSO peak). LCMS (m/z) [M+H] 695.0.

2) 5-[[2,5-Dihydroxy-4-[[4-hydroxy-3-(2-morpholinoethoxycarbonyl)phenyl]carbamoyl] benzoyl] amino]-2-hydroxy-benzoic acid (Ic-007a-mpe1)

Esterification. A solution of 5-[[2,5-dibenzyloxy-4-[(3-carboxy-4-hydroxyphenyl)-carbamoyl]benzoyl]amino]-2-hydroxy-benzoic acid (324 mg, 0.5 mmol) and triethylamine (101 mg, 0.15 mL, 1 mmol) in N,N-dimethylformamide (7 mL) was stirred at room temperature. A solution of 2-morpholinoethanol (66 mg, 0.5 mmol) in N,N-dimethylformamide (1 mL) and N,N-dimethylpyridin-4-amine (122 mg, 1 mmol) was added. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (115 mg, 0.6 mmol) was added and the reaction mixture was stirred at room temperature for 2 hours and then heated in a microwave oven at 60 °C for 4 hours. The reaction was repeated on a similar scale and the reaction mixtures were combined. The solvent was evaporated and the residue was triturated with diethyl ether to give a mixture of mono and diester (230 mg). LCMS (m/z) [M+H] 762.0 (mono ester).

Hydrogenolysis. The above mono and diester mixture (230 mg) was dissolved in acetic acid (10 ml), tetrahydrofuran (15 ml) and water (5 ml) and 10% palladium on carbon (150 mg, 10% paste) was added. The reaction mixture was stirred under a hydrogen atmosphere for 16 hours. The catalyst was removed by filtration and the solvent was evaporated to give a yellow gum (140 mg). The residue was purified by reverse phase preparative HPLC on a C18 column using a gradient of 10-70% (acetonitrile 0.1% formic acid): (water 0.1% formic acid). The residue was triturated with diethyl ether and dried under vacuum to give the title monoester Ic-007a-mpe1 as a yellow solid (11 mg).

<1>H nmR (d6-DMSO ppm) δ 11.33-11.29 (br s, 1H), 11.13-10.08 (br s, 1H), 10.50-10.44 (br s, 2H), 10.36-10.30 (br s, 1H), 8.25 (d, J=2.4 Hz, 1H), 8.06-8.02 (m, 1H), 7.78 (dd, J=9.0, 2.4Hz, 1H), 7.69-7.60 (m, 2H), 7.52 (s, 1H), 7.01 (d, J=9.0 Hz, 1H), 6.75 (d, J=8.7 Hz, 1H), 4.50-4.45 (m, 2H), 3.65-3.55 (m, 4H), 2.81-2.71 (m, 2H), 2.60-2.50 (m, 4H, partially obscured by DMSO peak). LCMS (m/z) [M+H] 582.0.

3) 1-(2,2-dimethylpropanoyloxy)ethyl 5-[[4-[[3-[1-(2,2-dimethylpropanoyloxy)ethoxycarbonyl]-4-hydroxy-phenyl]carbamoyl]-2,5-dihydroxybenzoyl]amino]-2-hydroxy-benzoate (Ic-007a-pive2) and

4) 5-[[4-[[3-[1-(2,2-dimethylpropanoyloxy)ethoxycarbonyl]-4-hydroxyphenyl]carbamoyl]-2,5-dihydroxy-benzoyl]amino]-2-hydroxy-benzoic acid (Ic-007a-pive1)

(mixture of mono- and di-esters)

Esterification. A solution of 5-[[2,5-dibenzyloxy-4-[(3-carboxy-4-hydroxyphenyl)carbamoyl] benzoyl]amino]-2-hydroxy-benzoic acid (324 mg, 0.5 mmol) and triethylamine (152 mg 0.21 mmol) in N,N-dimethylformamide (10 m) was stirred at 0 °C and 1-iodoethyl 2,2-dimethylpropanoate (572 g) was added. mg, 2 mmol). The reaction mixture was allowed to warm to room temperature and stirred at room temperature for 24 hours. The dimethylformamide was evaporated and the residue partitioned between dichloromethane and aqueous sodium metabisulfite. The organic phase was dried over anhydrous magnesium sulfate and evaporated to give a 1:1 mixture of mono and diester (187 mg). The reaction was repeated and the 1:1 ester mixtures were combined. LCMS (m/z) [M+H] 777.0 (monoester) and 905.1 (diester).

(mixture of mono- and di-esters) (mixture of mono- and di-esters)

Hydrogenolysis

The mixture of mono and diester (320 mg) obtained above was dissolved in tetrahydrofuran (50 ml) and water (5 ml) and 10% palladium on carbon (300 mg, 10% paste) was added. The reaction mixture was stirred under a hydrogen atmosphere for 20 hours. The catalyst was removed by filtration and the solvent was evaporated. LCMS showed that only partial deprotection was achieved. The residue was dissolved in tetrahydrofuran (36 mL) and water (6 mL) and 10% palladium on carbon (600 mg, 10% paste) were added. The reaction mixture was stirred under a hydrogen atmosphere for 22 hours. The catalyst was removed by filtration and the solvent was evaporated. The residue was purified by reverse phase preparative HPLC on a C18 column, using a gradient of 40-97% (acetonitrile 0.1% formic acid): (water 0.1% formic acid). The residues were triturated with diethyl ether and dried under vacuum to give the title products:

Diester (Ic-007a-pive2): yellow solid (60 mg).<1>H nmR (d6-DMSO ppm) δ 11.05 (s, 1H), 10.46 (s, 1H), 10.18 (s, 1H), 8.18 (br s, 1H), 7.83-7.77 (m, 1H), 7.52 (s, 1H). 1H), 7.05-6.96 (m, 2H), 1.58 (d, J=5.5 Hz, 3H), 1.17 (s, 9H). LCMS (m/z) [M+H] 724.9.

Biological data: T1/2 (mouse plasma): 62.2 min (ester cleaved; 65% remaining after 1h); T1/2 (human plasma): > 180 min.

Monoester (Ic-007a-pive1): Yellow solid (35 mg).<1>H nmR (d6-DMSO ppm) δ 11.13 (s, 1H), 11.06 (s, 1H), 10.46 (s, 1H), 10.42 (s, 1H), 10.18 (s, 1H), 8.21-8.18 (m, 2H), 8.78-8.72 (m, 2H), 7.55 (s, 1H), 7.52 (s, 1H), 7.03-6.92 (m, 3H), 1.58 (d, J=5.5 Hz , 3H), 1.17 (s, 9H). LCMS (m/z) [M+H] 596.9

Biological data: T1/2 (mouse plasma): 44.6 min (ester cleaved; 49% remaining after 1h); T1/2 (human plasma): > 180 min.

Prodrugs IIc-007a

1) 2,2-dimethylpropanoyloxymethyl 5-[[3-[[3-(2,2-dimethylpropanoyloxymethoxycarbonyl)-4-hydroxy-phenyl]carbamoyl]-2,5-dihydroxybenzoyl]amino]-2-hydroxy-benzoate (IIc-007a-pivm2)

Esterification. A solution of 5-[[2,5-dibenzyloxy-3-[(3-carboxy-4-hydroxyphenyl)carbamoyl]benzoyl]amino]-2-hydroxy-benzoic acid (280 mg, 0.432 mmol) was prepared in N-dimethyl formamide and chloromethyl 2,2-dimethylpropanoate (137 µl, 0.95 mmol) was added, followed by triethylamine (167 µl, 1.2 mmol)) and sodium iodide. (143 mg, 0.95 mmol). The mixed solution was heated at 50 °C for 18 hours. The solution was then cooled and partitioned between saturated ammonium chloride solution and ethyl acetate. The organic layer was dried over sodium sulfate, filtered and evaporated. The residue was purified by silica column chromatography using a gradient of 100% iso-hexane to 50% ethyl acetate, 50% iso-hexane to give the diester as a colorless gum (192 mg, 51%). LCMS (m/z) [M+H] 876.9.

Hydrogenolysis: A solution of the diester obtained above (192 mg, 0.219 mmol) in tetrahydrofuran (4.5 mL) was prepared and added to a stirred suspension of 10% palladium on carbon (40 mg, 0.04 mmol) in water (1.5 mL). The stirred reaction mixture was placed under a hydrogen atmosphere for 18 hours. The reaction was filtered to remove the catalyst and the solution was evaporated to give a cream solid (142 mg). The solid was purified by HPLC on a C18 column using a gradient of 60% acetonitrile, 40% water to 100% acetonitrile (0.1% formic acid) to give the title diester IIc-007a-pivm2 as a white solid (75 mg, 49%).<1>H nmR (DMSO-D6 ppm) δ 13.03 (br s, 1H). 10.43 (br s, 2H), 10.16 (s, 2H), 9.52 (br s, 1H), 8.17 (d, J=2.7Hz, 2H), 7.82 (dd, J=8.9Hz, 2.7Hz, 2H), 7.55 (s, 2H), 7.02 (d, J=8.9Hz, 2H), 5.98 (s, 4H), 1.17 (s, 18H). LCMS (m/z) [MH] 694.9.

2) 5-[[3-[[3-[1-(2,2-dimethylpropanoyloxy)ethoxycarbonyl]-4-hydroxyphenyl]carbamoyl]-2,5-dihydroxy-benzoyl]amino]-2-hydroxy-benzoic acid (IIc-007a-pive1)

Esterification. A solution of 5-[[2,5-dibenzyloxy-3-[(3-carboxy-4-hydroxyphenyl)carbamoyl]benzoyl]amino]-2-hydroxy-benzoic acid (324 mg, 0.5 mmol) and triethylamine (101 mg, 0.14 ml, 1.0 mmol) in N,N-dimethylformamide (10 ml) was stirred at 0°C. 1-Iodoethyl 2,2-dimethylpropanoate (154 mg, 0.6 mmol) was added. The reaction mixture was stirred at room temperature for 21 hours. More triethylamine (0.14 mL, 1.0 mmol) and 1-iodoethyl 2,2-dimethylpropanoate (154 mg, 0.6 mmol) were added and the reaction mixture was stirred at room temperature for 1 hour. The solvent is evaporated. The residue was partitioned between dichloromethane and sodium metabisulfite solution. The organic extracts were combined, washed with saline and dried by passing the solution through a hydrophobic frit. The filtrate was evaporated to give a mixture of mono and diester. The residue was purified by silica gel chromatography eluting with 5-100% ethyl acetate in isohexanes followed by 10% methanol in dichloromethane to give the mono ester as a yellow solid (121 mg). LCMS (m/z) [M+H] 777.

Hydrogenolysis. A solution of the monoester obtained above (121 mg, 0.15 mmol) in tetrahydrofuran (16 ml) and water (4 ml) containing 10% palladium on carbon (250 mg, 10% paste) was stirred under a hydrogen atmosphere for 18 hours. The catalyst was removed by filtration and the solvent was evaporated. The residue was purified by preparative reverse phase HPLC using a gradient of 40-97% (acetonitrile 0.1% formic acid): (water 0.1% formic acid) to give IIc-007a-pive1 monoester as a yellow solid (34 mg ).<1>H nmR (DMSO-D6" ppm) δ 13.15 (br s, 1H), 10.47 (br s 2H), 10.18 (s, 1H), 9.47 (br s, 1H), 8.21 (d, J=3 Hz 1H), 8.17 (d, J=3 Hz 1H), 7.82 (dd, J=9, 3 Hz 1H), 7.78 (dd, J=9, 3 Hz 1H), 7.59-7.55 (m, 2H), 7.03 (d, J=9 Hz 1H), 6.99 (q, J=5.4 Hz 1H), 7.96 (d, J=9 Hz 1H), 1.58 (d, J=5.4 Hz , 3H), 1.17 (s, 9H). LCMS (m/z) [MH] 595.1.

Prodrugs IVc-059a

1) 2,5-dihydroxy-3-[[2,5-hydroxy-3-(2-morpholinoethoxycarbonyl)phenyl] methylsulfonyl-methyl]benzoic acid (IVc-059a-mpe1)

Esterification. A solution of 2,5-dibenzyloxy-3-[(2,5-dibenzyloxy-3-carboxyphenyl)methylsulfonylmethyl]benzoic acid (300 mg, 0.40 mmol) and 2-morpholinoethanol (53 mg, 0.40 mmol) was prepared in N,N-dimethylformamide (93 ml) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 93 mg) was added. 0.48 mmol) and N,N-dimethylpyridin-4-amine (10 mg, 0.08 mmol). The reaction mixture was stirred at room temperature for 18 hours. The resulting solution was evaporated to dryness and the residue was purified by HPLC on a C18 column using a gradient of 30% acetonitrile, 70% water to 100% acetonitrile (0.1% formic acid) to give the morpholinoethyl ester as a colorless gum (111 mg, 32%). LCMS (m/z) [M+H] 872.1.

Hydrogenolysis. A solution of the above monoester (111 mg, 0.127 mmol) in tetrahydrofuran (3 mL) was added to a stirred suspension of 10% palladium on carbon (20 mg, 0.019 mmol) in water (1 mL). The stirred reaction mixture was placed under a hydrogen atmosphere for 18 hours.

The reaction was filtered to remove the catalyst and the solution was evaporated to give a brown solid. The solid was purified by HPLC on a C18 column using a gradient from 10% acetonitrile, 90% water to 60% acetonitrile, 40% water (0.1% formic acid) to give a white solid (20.5 mg). The solid was triturated with diethyl ether and dried under vacuum to give the title product IVc-059a-mpe1 as a white solid (5 mg, 8%).

<1>H nmR (DMSO-d6 ppm) δ 10.33 (br s, 1H), 9.30 (s, 1H) 8.92 (s, 1H), 7.08-7.15 (m, 3H), 6.91 (d, J=3.1Hz, 1H), 4.40 (t, J=5.0Hz, 2H), 4.36 (s, 2H), 3.65-3.72 (m, 4H), 3.05 (t, J=5.0Hz, 2H), 2.78-2.87 (m, 4H). LCMS (m/z) [M+H] 511.9.

Biodata: stable up to 60 min in mouse plasma

2) 2,5-Dihydroxy-3-[[2,5-hydroxy-3-(2-morpholinoethoxycarbonyl)phenyl] methylsulfonyl-methyl]benzoic acid 2-morpholinoethyl ester (IVc-059a-mpe2)

A solution of 2,5-dibenzyloxy-3-[(2,5-dibenzyloxy-3-carboxyphenyl)methylsulfonylmethyl]benzoic acid (69 mg, 0.091 mmol) and 2-morpholinoethanol (25 mg, 0.19 mmol) was prepared in N,Ndimethylformamide (1 mL) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (42 mg, 0.48 mmol) was added. N,Ndimethylpyridin-4-amine (5 mg, 0.04 mmol). The reaction mixture was stirred at room temperature for 18 hours. The resulting solution was evaporated to dryness and the residue was purified by silica column chromatography using 10% methanol, 90% dichloromethane to give the title product as a colorless gum (58 mg, 65%). LCMS (m/z) [M+H] 985.0.

A solution of the above diester (58 mg, 0.059 mmol) in tetrahydrofuran (1.5 mL) was prepared and added to a stirred suspension of 10% palladium on carbon (5 mg, 0.005 mmol) in water (0.5 mL). The stirred reaction mixture was placed under a hydrogen atmosphere for 18 hours. The reaction was filtered to remove the catalyst and the solution was evaporated to give a brown solid. The solid was purified by HPLC on a C18 column using a gradient from 10% acetonitrile, 90% water to 60% acetonitrile, 40% water (0.1% formic acid) to give a white solid (13.7 mg).

The solid was triturated with diethyl ether and dried under vacuum to give the title product IVc-059a-mpe2 as a white solid (9.1 mg, 25%).

<1>HNMR (CD3OD ppm) δ 8.34 (brs, 2H), 7.32 (d, J=3.1Hz, 2H), 7.16 (d, J=3.1Hz, 2H), 4.52 (t, J=5.5Hz, 4H), 4.45 (s, 4H), 3.68-3.73 (m, 8H), 2.83 (t, J=5.5Hz, 4H), 2.58-2.65 (m, 8H). LCMS (m/z) [M+H] 625.0.

Biodata: stable up to 60 min in mouse plasma; T1/2 (human

plasma): > 180 min.

3) 3-[[3-[1-(2,2-dimethylpropanoyloxy)ethoxycarbonyl]-2,5-dihydroxyphenyl]methyl-sulfonylmethyl]-2,5-dihydroxy-benzoic acid 1-(2,2-dimethylpropanoyloxy) )ethyl ester (IVc-059a-pive2)

Esterification. A solution of 2,5-dibenzyloxy-3-[(2,5-dibenzyloxy-3-carboxyphenyl)methyl-sulfonylmethyl]benzoic acid (200 mg, 0.263 mmol) in anhydrous N,N-dimethylformamide (5 ml) was prepared and triethylamine (88 µl, 0.63 mmol), sodium iodide (8 mg, 0.053 mmol) and crude 1-iodoethylamine were added. 2,2-dimethylpropanoate (ca. 4.2 mmol) was added. The reaction mixture was stirred at room temperature for 18 hours. The resulting solution was evaporated to a small volume and the residue was partitioned between dichloromethane and sodium thiosulfate solution. The organic layer was filtered through a hydrophobic frit and evaporated. The residue was purified by silica gel column chromatography eluting with a gradient from 100% iso-hexane to 50:50 ethyl acetate/iso-hexane to give the di-ester as a colorless gum (110 mg, 41%). LCMS (m/z) [M+Na] 1037.0

The monoester was also isolated from the same column as the colorless gum (90 mg, 40%). LCMS (m/z) [M+Na] 909.6.

Hydrogenolysis:

A solution of the diester isolated above (110 mg, 0.108 mmol) in tetrahydrofuran (3 mL) was added to a stirred suspension of 10% palladium on carbon (20 mg, 0.02 mmol) in water (1 mL). The stirred reaction mixture was placed under a hydrogen atmosphere for 48 hours. The reaction mixture was filtered to remove the catalyst, the solution was evaporated, and the residue was purified by silica column chromatography eluting with a gradient of 100% dichloromethane to 10% methanol, 90% dichloromethane to give the title product IVc-059a-pive2 as a solid (55 mg, 78%).

<1>H nmR (CDCl3 ppm) δ 10.55 (s, 2H), 7.24-7.28 (m, 2H), 7.14-7.18 (m, 2H), 7.05 (q, J=5.4Hz, 2H), 6.18 (br s, 2H), 4.30-4.50 (m, 4H), 1.60 (d, J=5.4Hz, 6H), 1.21 (s, 18H). LCMS (m/z) [MH] 653.9.

Biodata: rapidly hydrolyzed in mouse and human plasma

4) 3-[[3-[1-(2,2-dimethylpropanoyloxy)ethoxycarbonyl]-2,5-dihydroxyphenyl]methylsulfonyl-methyl]-2,5-dihydroxy-benzoic acid (IVc-059a-pive1)

Hydrogenolysis:

A solution of the monoester isolated above (90 mg, 0.104 mmol) in tetrahydrofuran (3 mL) was added to a stirred suspension of 10% palladium on carbon (20 mg, 0.02 mmol) in water (1 mL). The stirred reaction mixture was placed under a hydrogen atmosphere for 48 hours. The reaction was filtered to remove the catalyst, the solution was evaporated and the residue was purified by silica column chromatography eluting with a gradient of 100% dichloromethane to 20% methanol, 80% dichloromethane to give the title product IVc-059a-pive1 as a solid (22.5 mg, 43%).

1H nmR (CD3OD ppm) δ 7.39 (d, , J=3.1Hz, 1H), 7.25 (d, J=3.1Hz, 1H), 7.18 (d, J=3.1Hz, 1H), 7.05 (q, J=5.4Hz, 1H), 6.96 (d, J=3.1Hz, 1H), 4.40-4.45 (m, 4H), 1.61 (d, J=5.4Hz, 3H), 1.21 (s, 9H). LCMS (m/z) [MH] 524.9.

Biodata: rapidly hydrolyzed in mouse plasma

5) 2,2-dimethylpropanoyloxymethyl 2,5-dihydroxy-3-[[2,5-dihydroxy-3-(2,2-dimethyl-propanoyloxymethoxycarbonyl)phenyl]methylsulfonylmethyl] benzoate (IVc-059a-pivm2)

Esterification. A solution of 2,5-dibenzyloxy-3-[(2,5-dibenzyloxy-3-carboxyphenyl)methylsulfonyl-methyl]benzoic acid (100 mg, 0.131 mmol) in anhydrous N,N-dimethylformamide (1 ml) was prepared and chloromethyl 2,2-dimethylpropanoate (91 µl, 0.631 mmol) was added followed by triethylamine (110 µl, 0.631 mmol). 0.789 mmol) and sodium iodide (87 mg, 0.579 mmol). The reaction mixture was heated at 50 °C for 6.5 hours. The resulting solution was evaporated, and the residue was partitioned between ethyl acetate and water. The organic layer was dried over sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography eluting with a gradient of 100% iso-hexane to 50% ethyl acetate, 50% iso-hexane to give the diester product as a colorless gum (85 mg, 66%). LCMS (m/z) [M+Na] 1010.0.

Hydrogenolysis: A solution of the diester obtained above (85 mg, 0.086 mmol) in tetrahydrofuran (3 mL) was added to a stirred suspension of 10% palladium on carbon (20 mg, 0.02 mmol) in water (1 mL). The stirred reaction mixture was placed under a hydrogen atmosphere for 18 hours. The reaction was filtered to remove the catalyst, the solution was evaporated, and the residue was purified by silica gel column chromatography eluting with a gradient of 100% isohexane to 60% ethyl acetate, 40% isohexane to afford diester IVc-059a-pivm2 as a white solid (31 mg, 57%).

<1>H nmR (CDCl3 ppm) δ 7.29 (d, J=2.9Hz, 2H), 7.19 (d, J=2.9Hz, 2H), 5.97 (s, 4H), 4.42 (s, 4H), 1.22 (s, 18H). LCMS (m/z) [MH] 624.8.

Biodata: rapidly hydrolyzed in mouse plasma, T1/2 = 14.1 min; T1/2 (human plasma): > 180 min.

6) 2,5-dihydroxy-3-[[2,5-dihydroxy-3-(2,2-dimethylpropanoyloxymethoxycarbonyl)phenyl] methyl-sulfonylmethyl] benzoic acid (IVc-059a-pivm1)

Esterification: A solution of 2,5-dibenzyloxy-3-[(2,5-dibenzyloxy-3-carboxyphenyl)methylsulfonyl methyl]benzoic acid (300 mg, 0.395 mmol) in anhydrous N,N-dimethylformamide (3 ml) was prepared and chloromethyl 2,2-dimethylpropanoate (57 µl, 0.395 mmol) was added followed by triethylamine (68 µl, 0.395 mmol). 0.489 mmol) and sodium iodide (129 mg, 0.870 mmol). The reaction mixture was heated at 50 °C for 4 hours. The resulting solution was cooled and then evaporated. The residue was purified by silica column chromatography eluting with a gradient of 20% ethyl acetate, 80% isohexane to 80% ethyl acetate, 20% isohexane to give the monoester product as a white solid (121 mg, 35). %). LCMS (m/z) [MH] 871.0.

Hydrogenolysis: A solution of the monoester obtained above (121 mg, 0.138 mmol) in tetrahydrofuran (3 mL) was added to a stirred suspension of 10% palladium on carbon (20 mg, 0.02 mmol) in water (1 mL). The stirred reaction mixture was placed under a hydrogen atmosphere for 48 hours. The reaction was filtered to remove the catalyst, the solution was evaporated and the residue was purified by HPLC on a C18 column eluting with a gradient of 30% acetonitrile, 70% water to 100% acetonitrile (0.1% formic acid) to give monoester IVc-059a-pivm1 as a white solid (30 mg, 42%).

<1>H nmR (CD3OD ppm) δ 7.34 (d, J=3.0Hz, 1H), 7.27 (d, J=3.1Hz, 1H), 7.20 (d, J=3.1Hz, 1H), 7.08 (d, J=3.0Hz, 1H), 6.01 (s, 2H), 4.45 (s, 4H), 1.22 (s, 9H). LCMS (m/z) [MH] 510.9.

Biodata: rapidly hydrolyzed in mouse plasma, T1/2 = 6.7 min; T1/2 (human plasma): > 180 min.

EXAMPLE 2: General experimental procedures

NMR spectroscopy

<1>H nmR spectra were recorded at 400 MHz on a Bruker Advance III nmR spectrometer. Samples were prepared in deuterated chloroform (CDCl3) or dimethylsulfoxide (DMSO-d6) and raw data were processed using Mnova nmR software. High-resolution mass spectra were recorded with a MaXis ESI qTOF ultra-high-resolution mass spectrometer

UPLC-MS analysis

UPLC-MS analysis was performed on a Waters Acquity UPLC system consisting of an Acquity I-Class Sample Manager-FL, an Acquity I-Class Binary Solvent Manager and an Acquity I-Class UPLC Column Manage. UV detection was achieved using an Acquity I-Class UPLC PDA detector (210 - 400 nm scan), while mass detection was achieved using an Acquity QDa detector (mass scan 100 - 1250 Yes; positive and negative modes simultaneously). A Waters Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 mm) was used to achieve analyte separation.

Samples were prepared by dissolving (with or without sonication) in 1 ml of a 1:1 (vol./vol.) mixture of MeCN in H2O. The resulting solutions were filtered through a 0.45 µm syringe filter before being submitted for analysis. All solvents used (including formic acid and 36% ammonia solution) were of HPLC grade.

Four different analytical methods were used for this work, the details of which are presented below. Acid run (2 min): 0.1% vol./vol. Formic acid in water [Eluent A]; 0.1% vol./vol. Formic acid in MeCN [Eluent B]; Flow rate 0.8 ml/min; injection volume 2 µl and equilibration time between samples 1.5 min.

Table 14:

Acid run (4 min): 0.1% vol./vol. Formic acid in water [Eluent A]; 0.1% vol./vol. Formic acid in MeCN [Eluent B]; Flow rate 0.8 ml/min; injection volume 2 µl and equilibration time between samples 1.5 min.

Table 15:

Acid run (6.5 min): 10 mm ammonium formate 0.1% vol./vol. formic acid [Eluent A]; 0.1% vol./vol. Formic acid in MeCN [Eluent B]; Flow rate 0.6 ml/min; Injection volume 2 µl.

Table 16:

Basic run (2 min): 0.1% ammonia in water [Eluent A]; 0.1% ammonia in MeCN [Eluent B]; Flow rate 0.8 ml/min; injection volume 2 µl and equilibration time between samples 1.5 min.

Table 17:

Basic run (4 min): 0.1% ammonia in water [Eluent A]; 0.1% ammonia in MeCN [Eluent B]; Flow rate 0.8 ml/min; injection volume 2 µl and equilibration time between samples 1.5 min.

Table 18:

Basic run (6.5 min): 10 mm ammonium bicarbonate 0.1% vol./vol. 35% ammonia solution [Eluent A]; 0.1% vol./vol. Formic acid in MeCN [Eluent B]; Flow rate 0.6 ml/min; Injection volume 2 µl.

Table 19:

Preparative HPLC purification

Preparative HPLC was performed using a Waters autopurification system with an XBridge Prep C18 column (19 × 150 mm). The Waters system consisted of a Waters 2545 Binary Prep pump, a Waters 2767 Sample Manager, a Waters SFO Column Organizer, a Waters 2998 DAD, a Waters 515 Make up pump and an Acquity QDa mass spectrometer

The purification system is controlled by MassLynx software (version 4.2) with Open Access Login module.

The mobile phases consisted of (A) 10 mm ammonium formate (+ 0.1% vol./vol. formic acid) and (B) acetonitrile (+ 0.1% vol./vol. formic acid).

The samples were prepared by dissolving in 10% vol./vol. of water in DMSO to give a concentration of ~150 mg/ml. A volume of 320 ml of the prepared sample was loaded onto the column and purified using gradient elution at room temperature with a flow rate of 25 ml/min. There is a balance of 1.5 minutes between injections.

Table 20: Gradient:

EXAMPLE 3: In vitro screening methods

Example 3.1: In vitro method of inhibition of HUVEC cells by FGF1, FGF2 and VEGF-A

The aim of this in vitro method is to evaluate the effects of compounds on FGF-2, FGF-1 or VEGF-A induced sprouting of human umbilical vein endothelial cells (HUVEC) in a spheroid-based cell angiogenesis assay. Before adding the growth factor to the cells, they were pre-incubated with the compound. Sunitinib was tested as a control (no preincubation).

Control sunitinib inhibited HUVEC sprouting induced by FGF-2, FGF-1 and VEGF-A as expected. The IC50 values determined for the tested compounds are expressed in microM [µM], and the values for inhibition of growth (sprouting) induced by FGF-1, FGF2 and VEGF-A are listed after each compound in the examples after chemical characterization.

A germination assay was performed on compounds dissolved in DMSO to achieve a 100-fold concentrated stock solution and dissolved in culture medium. Sunitinib as a reference compound was provided by ProQinase GmbH. Growth factors were purchased from rhFGF-basic (FGF-2), Peprotech, New Jersey, USA, #100-18C, Lot # 0415571; rhFGF-acidic (FGF-1), Peprotech, New Jersey, USA, # 100-17A, Lot # 031207; hVEGF-A165 (ProQinase GmbH, Freiburg, Germany;

02/05/2010) were used to stimulate the growth of HUVEC cells.

Test System: Human umbilical vein endothelial cells (HUVEC) collected from donors were stored frozen with 70% medium, 20% FCS, 10% DMSO at about 1 × 10<6>cells/ampoule. HUVEC cells, primary human umbilical vein endothelial cells (PromoCell, Heidelberg, Germany), were used after passages 3 to 4. Cell morphology is adherent, growing as a cobblestone-like monolayer, and cultured in endothelial cell growth and basal medium (ECGM/ECBM, PromoCell). The subculture was divided 1:3; every 3-5 days, seed at ca. 1 × 10<4>cells/cm<2>i incubated at 37 °C with 5% CO2 with a doubling time of 24-48 hours

Dilution of test compounds: 100x concentrated solutions (relative to the final test concentrations) of each test compound were prepared with the appropriate solvent (in DMSO or in water). These 100x compound solutions were further diluted in endothelial cell growth and basal medium (ECBM) 1:7.5 to obtain a 13.33x concentrated solution. 75 µl of the 13.33x solution was then mixed with 25 µl of 40x concentrated stimulus (dissolved in ECBM), resulting in a 10x concentrated stimulus/compound mixture. This mixture (100 µl) was incubated for 30 min at 37 °C and then added to 900 µl of gel containing HUVEC spheroids (resulting in the final concentration of the assay).

Test Procedure: Experiments were performed in a modification of the originally published protocol (Korff and Augustin: J Cell Sci 112: 3249-58, 1999). Briefly, spheroids were prepared as described (Korff and Augustin: J Cell Biol 143: 1341-52, 1998) by pipetting 400 HUVECs in a hanging drop onto plastic dishes to allow spheroid aggregation overnight. 50 HUVEC spheroids were then seeded into 0.9 ml collagen gel and pipetted into individual wells of a 24-well plate to allow polymerization. Test compounds were pre-incubated with growth factors (FGF-1, FGF-2 or VEGF-A final concentration 25 ng/ml) in a 10-fold concentrated solution, and then 100 µl of this solution was added to the polymerized gel solution. All compounds were diluted in solvent (DMSO or water) as first 100x stocks (meaning 100x stocks of each final assay concentration were prepared in solvent). Subsequently, these stocks were further diluted in medium (resulting in 13.33x stocks of each final assay concentration) with final concentrations tested: 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 × 10<-6>M). Plates were incubated at 37 °C for 24 h and fixed by adding 4% PFA (Roth, Karlsruhe, Germany).

Quantification of growth factor inhibition: The sprouting intensity of HUVEC spheroids treated with growth factor and inhibitors was quantified using an image analysis system that determines the cumulative sprout length per spheroid (CSL). Images of individual spheroids were captured using an inverted microscope and NIS-Elements BR 3.0 digital imaging software (Nikon). After that, the spheroid images were uploaded to the Wimasis home page for image analysis. The cumulative sprout length of each spheroid was determined using the WimSprout image analysis tool.

The mean cumulative germ length of 10 randomly selected spheroids was analyzed as a single data point. For IC50 determination, raw data were converted to percent HUVEC germination relative to the solvent control (containing stimulus), which was set to 100%, and the basal control (no stimulation), which was set to 0%. IC50 values were determined using GraphPad Prism 5 software with a bottom limit of 0 and a peak limit of 100 using a non-linear regression curve with a variable slope of the hill.

Results: A dose-response relationship for growth factor-induced endothelial cell sprouting was assessed for all compounds. IC50 values were determined using standard parameters based on the solvent control signal as the upper limit (100% HUVEC germination) and the basal control signal as the lower limit (0%). The corresponding IC50 values for each compound on FGF1, FGF2 and VEGFA are summarized below each compound below their chemical characterization in the BIODATA paragraph.

Example 3.2: In vitro adhesion of neutrophils in static conditions, method

Human neutrophils obtained from the fovea of healthy donors were suspended at 3 × 10<6>/ml in adhesion buffer (PBS containing 1 mm CaCl2/MgCl2 and 10% FCS, pH 7.2). Neutrophils were pre-incubated for 30 min at 37 °C with 40 µm and 80 µm compounds.

Adhesion tests were performed on 18-well glass slides coated for 18 h at 4 °C with human fibrinogen (20 µg/well in PBS without endotoxin); 20 µl of cell suspension was added to each well and stimulated for 1 min at 37 °C with 5 µl of a final concentration of fMLP 1nM. After washing, adherent cells were fixed in glutaraldehyde 1.5% in PBS and counted by computer-assisted counting. Statistical analysis was performed by calculating the mean value and standard deviation (SD); significance was calculated by unpaired t-test. All statistical analyzes were performed using GraphPad Prism 6 (GraphPad Software).

The corresponding inhibition of neutrophil adhesion expressed in [%] for each compound is summarized below each compound below their chemical characterization in the BIODATA paragraph.

Example 3.3: In vitro testing of inhibition of reactive oxygen species (ROS) production in human neutrophils

NADPH oxidase, located in the plasma membrane and in the membranes of specific granules, produces superoxide anions from which ROS remain, such as hydrogen peroxide, singlet oxygen, hypochlorite and active hydroxyl radicals. ROS are released into the surrounding medium or into a membrane-enclosed subcellular organelle. One rapid and sensitive method for measuring the formation of these metabolites is chemiluminescence (CL). The isoluminol-enhanced CL technique is very sensitive and widely used to study the respiratory burst (mainly extracellular ROS production) induced in neutrophils.

Human neutrophils were isolated from reptilian coats obtained from healthy donors. Isolated neutrophils (3 × 10<6>/ml) were suspended in Hanks' balanced salt solution (HBSS) with calcium and magnesium. Neutrophils were pre-incubated with compounds at different concentrations (0.1, 0.3, 1, 3 and 10 µm) for 30 minutes at 37 °C. Black 96-well cell culture plates were coated with human fibrinogen (0.25 mg/ml in PBS) and incubated overnight at 4 °C or 2 hours at 37 °C. The plates were then washed with endotoxin-free PBS.

Neutrophils were incubated (priming) with TNF-α (20 ng/ml) for 10 minutes at 37 °C. Then 75 µl of cell suspension and 75 µl (isoluminol HRP solution) were added to each well. Chemiluminescence was recorded (every 25 seconds for 250 seconds) after fMLP (1 µm) activation using a Multilabel Reader victor3 (Perkin Elmer, USA). Background values, defined as the mean chemiluminescence values of isoluminol diluted in HBSS, were subtracted from all readings (CPS, counts per second). Tests were performed in duplicate or triplicate.

The results obtained from the isoluminol-enhanced CL assay revealed that the compounds strongly reduced the level of ROS in a concentration-dependent manner in fMLP TNF-α stimulated PMN.

Inhibition of PMN ROS at 0.3 µm expressed in [%] and IC50 [µM] are summarized below each compound below their chemical characterization in the BIODATA paragraph.

Example 3.4: In vitro production of human neutrophils and human monocytes of inflammatory cytokines, procedure

Neutrophils and monocytes were isolated from downy coats of healthy donors, under endotoxin-free conditions. Briefly, foam coatings were stratified on a Ficoll-Paque PLUS gradient and then centrifuged at 400 × g for 30 min at room temperature. Neutrophils were then collected and subjected to dextran sedimentation followed by hypotonic lysis to remove erythrocytes. Instead, monocytes were isolated from PBMC, after centrifugation over a Percoll gradient. Human neutrophils and monocytes were suspended at 5×106/ml and 2.5×106/ml, respectively, in RPMI 1640 medium supplemented with 10% low-endotoxin FBS (<0.5 EU/ml; from BioWhittaker-Lonza, Basel, Switzerland) and then plated in 24-well tissue culture plates at 37 °C, 5% CO2, in the presence or in the absence of different concentrations (20-40 µm) of compounds. After 1 h of drug treatment, monocytes were stimulated with 0.1 µg/ml, while neutrophils with 1 µg/ml ultrapure LPS from E. coli 0111:B4 strain (InvivoGen). After 6 h of LPS stimulation, neutrophils and monocytes were collected and centrifuged at 300 × g for 5 min. Cell-free supernatants were immediately frozen at -80 °C.

Cytokine concentrations in cell-free supernatants were measured with commercially available human-specific ELISA kits: CXCL8, IL-6, TNF-α (Mabtech, Sweden). The detection limits of these ELISAs were: 4 pg/ml for CXCL8, 10 pg/ml for IL-6 and 4 pg/ml for TNF-α.

Statistical analysis was performed by calculating the mean value and standard deviation (SD); significance was calculated by unpaired t-test. All statistical analyzes were performed using GraphPad Prism 6 (GraphPad Software).

Cytokine values are expressed in ng/ml for each compound and summarized for each compound below their chemical characterization in the BIODATA section.

Example 3.5: In vitro inhibition by compounds of VEGF-165 induced phosphorylation of VEGF-receptor on primary umbilical vein endothelial cells (HUVEC), procedure

Cell culture: Primary umbilical vein endothelial cells (HUVEC) were routinely cultured in 0.1% gelatin pre-coated flasks or dishes, up to passage 6. The effect of the compounds themselves at various concentrations (0, 1.25, 2.5, 5, 10 and 20 µm) on cell viability was assessed with a CCK-8 kit using a Tecan Microplate Reader (Genios). To measure the effect of compounds on VEGF-induced cell viability, HUVECs (1 × 10<4>cells/well) were treated with VEGF (25 ng/ml) premixed with different concentrations of compounds (0, 1.25, 2.5, 5, 10 and 20 µm) in culture medium without serum and growth factors (starvation medium) for 24 h and 48 h. Compounds in this concentration range did not affect cell viability.

Rabbit primary antibodies for VEGFR-2, p-Tir1175 were purchased from Cell Signaling Technology (Leiden, The Netherlands). Phospho-VEGFR-2 (Tir1175) sandwich ELISA kit was from Cell Signaling Technology.

HUVECs were pre-incubated with compounds at 0, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µm for 60 min with three subsequent washing steps with warm medium before stimulation with VEGF165 (25 ng/ml). Western blot analysis was performed using anti-phospho-VEGFR-2 antibody, and total VEGFR-2 was used as a loading control after membrane stripping.

Results of inhibition of VEGFR-2-phosphorylation are expressed as IC50 in microM [µM] relative to VEGF-treated HUVEC control without inhibitory compound.

The corresponding IC50 values expressed in [µM] for each compound are summarized below each compound below their chemical characterization in the BIODATA paragraph.

Example 3.6: In Vitro Inhibition of Cytokine Release by Compounds Following LPS Induction in Human Whole Blood

The aim of this in vitro method is to evaluate the effects of compounds on the release of a panel of cytokines from whole blood following lipopolysaccharide (LPS)-induced inflammatory response. Whole blood was incubated with compounds for 1 h before LPS induction. Blood cytokine levels were measured using a multiplex FACS-based panel.

IC50 values determined for the tested compounds expressed in microM [uM] and values for cytokine inhibition are listed for each compound.

The effect on cytokine release after LPS induction was performed on compounds dissolved in DMSO or H2O to achieve a 10-fold concentrated stock solution and dissolved in the culture medium.

Test System: Whole blood was removed from one donor (into lithium heparin-coated tubes; BD Vacutainer; 367886) and diluted 1:5 with RPMI 1640 (Thermo Fisher, 61870036). Within 30 min of sampling, blood was incubated with compounds for 1 h before LPS stimulation (O111:B4 - Sigma L4391; 10 ng/ml final concentration). Stimulation was continued under standard cell culture conditions (37 °C with 5% CO2) overnight (18 h), after which the plates were centrifuged and the supernatant removed and frozen at -80 °C until used for analyses.

Dilution of test compounds: 10x concentrated solutions (relative to the final test concentrations) of each test compound were prepared with the appropriate solvent (in DMSO or in water). These 10× compound solutions (10 µl) were then added to 90 µl of prediluted whole blood (whole blood: RPMI1640, 1:5) resulting in the final concentration of the assay.

Test procedure: Diluted whole blood (80 µl) was preincubated with compounds (10 µl) to obtain final test concentrations of 100, 30, 10, 3, 1, 0.3 and 0.1 × 10<-6>M. Blood was incubated at 37 °C with 5% CO2 for 1 hour. LPS was diluted in cell culture medium to a concentration of 110 ng/ml; 10 µl was added to whole blood to give a final concentration of 10 ng/ml. The plates were incubated at 37 °C for 18 h. Plates were centrifuged at 3000 rpm for 5 min at 4 °C, and the supernatant was removed and frozen at −80 °C until FACS analysis was performed.

Cytokine release was measured using a custom multiplex system (ELISA Genie) according to the manufacturer's instructions. Prepared samples were run on an Attune NkT flow cytometer (Thermo Fisher).

Quantification of inhibition of cytokine release: known concentration standards were used as part of the cytokine panel to allow quantification of each cytokine concentration in each sample. This was performed using FCAP Array v3.0 software. For IC50 determination, raw data were converted to percentage concentrations relative to the solvent control, which was set to 100%. IC50 values were determined using GraphPad Prism 8 software with a bottom limit of 0 and a peak limit of 100 using a non-linear regression curve with a variable slope of the hill.

Results: A dose response relationship to cytokine release following LPS induction of an inflammatory response in whole blood was assessed for all compounds. IC50 values were determined for each compound for a panel of cytokines (GM-CSF, IFN-gamma, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p40, IL-13, IL-17A, IL-17F, IL-18, IL-21, IL-22, IL-33, TGF-beta, TNF-alpha, TNF-beta). The corresponding cytokine IC50s expressed in [µM] values for each compound are summarized below each compound under their chemical characterization in the BIODATA paragraph and also in Table 13.

EXAMPLE 4: Compound Formulations

Example 4.1: Oral Formulations of Compound Ic-007a

Five formulations were developed and reviewed for suitability. These were both suspensions and solutions whose details are given in the following table.

Table 21: Formulation details for suspension and solutions of compound Ic-007a.

Production: Suspension A and solution D:

Example method for 100 ml (scale as needed for final volume). A solvent stock was prepared by measuring 5 ml of DMSO into a clean container. The exact amount of compound Ic-007a was measured and gradually added to the DMSO solution with mixing on a shaker, vortex or sonicator. Solutol was melted in a water bath at 60 °C in a clean dry dish. As the solution solidified at room temperature, the molten Solutol was kept in a 60 °C water bath during formulation preparation. 10 g (10% vol./vol.) of molten Solutol was added to a heated suitably labeled (to a final volume of 100 ml) container. 10 ml of PEG 400 was added to Solutol in a heated 100 ml container. DMSO stock solution of the drug was added to the Solutol and PEG400 mixture and the contents were immediately mixed, then 35 ml of deionized water was added and mixed in a 40 °C bath with occasional vortexing to dissolve large particles. The formulation was cooled to room temperature and made up to a final volume of 100 ml with deionized water.

Production of compounds A, B and C:

Example method for 100 ml (scale as needed for final volume). The solvent stock was prepared by measuring 5 ml of DMSO into a clean container. 1 g of compound Ic-007a was weighed and gradually added to the DMSO solution with mixing on a shaker, vortex or sonicator if necessary). Solutol was melted in a water bath at 60 °C in a clean dry dish. As Solutol solidifies at room temperature, molten Solutol was kept in a 60 °C water bath during formulation preparation. 10 g (10% vol./vol.) of molten Solutol was weighed into a heated, suitably labeled (to a final volume of 100 ml) container. 10 ml of PEG 400 was added to Solutol in a heated 100 ml container. DMSO drug stock solution was added to a labeled container of Solutol and PEG 400 mixture and immediately mixed with the contents. 35 ml of deionized water was added and mixed with the contents and placed in a bath at 40 °C with occasional vortexing to dissolve large particles. The formulation was cooled to room temperature and the pH of the formulation was further adjusted using 1M sodium hydroxide (NaOH) solution to pH 6, 7, 8 and 9. Initial formulations were made on a 1 ml scale and had an initial pH of 4.3. A volume of 3 µl NaOH 1M was added to achieve pH 6 (solution A), 22 µl for pH 8 (solution B) and 43 µl for pH 9 (solution C). These formulations are dosed immediately after production.

The 10 mg/ml suspension formulation and the 1 mg/ml solution formulation were found to be physically stable for two weeks at ambient conditions.

Example 4.2: Oral Formulations of Compound Ia-001a-Tz

Compound Ia-001a-Tz was prepared as a 10 mg/ml oral solution in formulations with the carrier compositions shown in the table below.

A vehicle was a carrier or inert medium used as a solvent (or diluent) in which the medicinally active agent was formulated and/or administered.

Table 22: Formulation details for the oral formulation of compound Ia-001aTz.

Production process

To prepare a 10 mg/g solution, 10 mg of compound was accurately weighed into a vial and 1 ml of vehicle was added directly to the API and vortexed to obtain a solution.

Vehicle preparation

Formulation A: 10 g of carrier was prepared by weighing 4 g of PEG400 into a clean 20 ml glass container. 1 g of Transcutol was added and the components were mixed by stirring. To this was added 1 g of Solutol HS15 followed by 4 g of Type 1 water. The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Formulation B: 10 g of carrier was prepared by weighing 4 g of PEG400 into a clean 20 ml glass container. 1 g of Transcutol was added and the components were mixed by stirring. To this was added 1 g of Solutol HS15.0.5% HPMC 606 aqueous solution was prepared by measuring 50 mg of HPMC 606 into a clean glass vial. To this was added 9.95 g of Type 1 water. The components were stirred for 1 h at room temperature to ensure adequate mixing. 4 g of an aqueous solution containing 0.5% HPMC 606 was then added to the solution of PEG400, Transcutol and Solutol HS15. The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Formulation C: 10 g of carrier was prepared by weighing 4 g of PEG400 into a clean 20 ml glass container. 1 g of Transcutol was added and the components were mixed by stirring. To this was added 1 g of Solutol HS15. Kollidon VA64 0.5% aqueous solution was prepared by measuring 50 mg of Kollidon VA64 into a clean glass vial. To this was added 9.95 g of Type 1 water. The components were mixed for 1 h at room temperature to ensure adequate mixing. 4 g of an aqueous solution containing Kollidon VA64 was then added to the solution of PEG400, Transcutol and Solutol HS15. The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve. Formulation D: 10 g of carrier was prepared by weighing 4 g of PEG400 into a clean 20 ml glass container. 1 g of Transcutol was added and the components were mixed by stirring. To this was added 1 g of Cremophor RH40 followed by 4 g of Type 1 water. The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Formulation E: 10 g of carrier was prepared by weighing 4 g of PEG400 into a clean 20 ml glass container. 1 g of Transcutol was added and the components were mixed by stirring. To this was added 1 g of vitamin E TPGS followed by 4 g of Type 1 water. The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Formulation F: 10 g of carrier was prepared by weighing 4 g of PEG400 into a clean 20 ml glass container. 1 g of Transcutol was added and the components were mixed by stirring. To this was added 0.5 g of Solutol HS15 followed by 4.5 g of Type 1 water. The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Formulation G: 10 g of carrier was prepared by weighing 4 g of PEG400 into a clean 20 ml glass container. 1 g of Transcutol was added and the components were mixed by stirring. To this was added 0.5 g of Solutol HS15. A 1% aqueous solution of Kollidon VA64 was prepared by measuring 100 mg of Kollidon VA64 into a clean glass vial. To this was added 9.9 g of Type 1 water. The components were mixed for 1 h at room temperature to ensure adequate mixing. 4.5 g of an aqueous solution containing Kollidon VA64 was then added to a solution of PEG400, Transcutol and Solutol HS15. The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Example 4.3. Oral formulations of compounds IIc-007a, IVc-059a, IIIc-061a

Compounds IIc-007a, IVc-059a, IIIc-061a were prepared as oral formulations using the components listed in the table below.

Table 23: Oral Formulation Details for Oral Formulation of Compounds IIc-007a, IVc-059a, IIIc-061a

Preparation of an aqueous solution of SLS 15% on a scale of 50 ml:

7.5 g of SLS was accurately measured in a 50 ml volumetric flask.

Water was added to a volume of 50 ml and mixed until complete solubilization was achieved.

Preparation of means for formulation:

Formulation 1: 100 g of carrier was prepared by weighing 40 g of PEG 400 (40.0%) into a plastic beaker. 3.5 g of Transcutol (3.5%), 12.5 g of Cremophor RH40 (12.5%), 4.0 g of Meglumine (4.0%), 5 g of DMSO (5.0%) and 35 g of 15% SLS water. solution (35.0%, final SLS 5.25%). The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Formulation 2: 100 g of carrier was prepared by weighing 40 g of PEG 400 (40.0%) into a plastic cup. 10.0 g of Transcutol (10.0%), 15.0 g of Cremophor RH40 (15.0%), 4.0 g of Meglumine (4.0%), 5 g of DMSO (5.0%) and 35 g of 15% SLS water. solution (35.0%, final SLS 5.25%). The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Vehicle was added directly to each compound before vortexing and sonication for 10 min. If necessary, the pH of the formulation is adjusted with 1M sodium hydroxide or 1M hydrochloric acid to reach a pH range suitable for oral dosing. Solutions of IIc-007a were made at concentrations up to and including 70 mg/ml. Solutions of IIIc-061a were made at concentrations up to and including 40 mg/ml. IVc-059a solutions were made at concentrations up to and including 150 mg/ml.

Example 4.4: Intravenous (IV) Formulations

Compounds Ia-001a, Ia-001a-Tz, Ia-001a-Tz/004a, Ic-007a, Ib-010a, IVc-059a, IIIc-061a were prepared as intravenous formulations.

The formulation details given in the following Table 24 have been used for IV administration in various preclinical in vivo models. Solutions of each compound were prepared by adjusting the pH with sodium hydroxide solution until a solution was obtained.

Table 24: IV formulation composition

Production process:

Example procedure for 10 ml (scale as needed for final volume). The solvent stock was prepared by measuring 0.5 ml of DMSO into a clean container. 100 mg of each compound was weighed and gradually added to the DMSO solution with mixing on a shaker, vortex or sonicator if necessary. Solutol was melted in a water bath at 60 °C in a clean dry dish. As Solutol solidified at room temperature, the molten Solutol was kept in a water bath at 60 °C during formulation preparation. 1 g (10% vol./vol.) of molten Solutol was weighed into a heated suitably labeled (to a final volume of 10 ml) container. 1 ml of PEG 400 was added to Solutol in a heated 10 ml container. DMSO drug stock solution was added to a labeled container of Solutol and PEG 400 mixture and immediately mixed with the contents. 0.9% saline* was added to reach 85% of the target volume and the contents were mixed in a 40 °C bath with occasional vortexing to dissolve large particles. The formulation was cooled to room temperature, made up to the final target volume (10 ml) with 0.9% saline* and the pH of the formulation was adjusted using 1M sodium hydroxide (NaOH) solution to pH 7.4. Formulations were filtered using a 0.2 µm filter into a sterile vial inside a Class II biological hood. The formulations were used for dosing in animals.

* For some compounds, the 0.9% saline was replaced with PBS pH 7.4.

Example 4.5: Intravenous (IV) and Intraperitoneal (IP) Formulations

Compounds Ic-007a, IIc-007a, IVc-059a, IIIc-061a were prepared as formulations for intravenous and intraperitoneal administration. The formulations contained water and diethylamine in molar equivalents of each respective compound as detailed in Table 25. These formulations were used for IV and IP administration in various preclinical in vivo models.

Production process:

Example procedure for 1 ml (scale as needed for final volume).

10 mg of each compound was accurately measured into a clean vial. The appropriate amount of diethylamine was added to the vial. Water was then added to achieve a volume of 1 ml. The contents are vortexed or sonicated to form a solution. Where necessary, the pH of the solution was adjusted using 1M hydrochloric acid to bring this into a range suitable for IV or IP dosing.

Table 25: molar equivalents of diethylamine required for compounds in IV and IP formulations.

Example 4.6: Ocular formulations

Several compounds of the present invention have been submitted for formulation development in an ophthalmic format. The compounds, their final concentration and form are given in the following Table 26.

Table 26: Ophthalmic formulations

Composition: The composition of the formulation is detailed in the following Table 27. The concentration of APIs and their resulting format were varied.

Table 27: Composition of the eye formulation.

Production process:

For those compounds that formed solutions, the following method of production was used:

A total of 50 mg of Kolliphor EL was transferred into a clean dry container. The exact amount of compound was measured and gradually added to Kolliphor EL with stirring on a magnetic stirrer. Heating to 30 °C helped to incorporate the drug into the Kolliphor preparation. In a separate container, an aqueous solution of HPMC at 0.5% vol./vol., povidone at 0.5% vol./vol. was prepared. and polysorbate 80 at 0.1% in pH 7.4 phosphate-buffered saline (PBS). A volume of 600 µl of the aqueous solution was added to the Kolliphor EL and drug during mixing and vortexed immediately. The pH was adjusted to 7.4 using 1 M NaOH in the formulation with stirring. The volume was adjusted to the target volume using an aqueous solution. The formulation was filtered using a 0.2 µm filter into a sterile vial inside a Class II biological hood. Formulations are ready for dosing.

For compounds that formed suspensions, the following production method was used: An amount of 50 mg of Kolliphor EL was measured into a clean dry container. The exact amount of different compounds was measured and gradually added to Kolliphor EL with stirring on a magnetic stirrer. Heating to 30 °C helped to incorporate the drug into the Kolliphor. In a separate container, an aqueous solution of HPMC at 0.5% vol./vol., povidone at 0.5% vol./vol. was prepared. and polysorbate 80 at 0.1% in pH 7.4 phosphate-buffered saline (PBS). The aqueous solution was filtered using a 0.22 µm filter. A volume of 600 µl of the aqueous solution was added to the Kolliphor and the drug while mixing and then immediately vortexed. The pH was adjusted to 7.4 using filtered 1 M NaOH with continuous mixing of the formulation. The volume was adjusted to the target volume using an aqueous solution. The formulations were sonicated prior to dosing to allow the flocculates to break up and disperse.

Ocular formulation of compounds Ic-007a, IIc-007a

Table 28: Composition of ocular formulation with Ic-007a and IIc-007a.

Production process:

A total of 50 mg of Kolliphor EL was transferred into a clean dry container. The exact amount of compound was measured and gradually added to Kolliphor EL with stirring on a magnetic stirrer. Heating to 30 °C helped to incorporate the drug into the Kolliphor preparation. In a separate container, an aqueous solution of HPMC at 0.5% vol./vol., povidone at 0.5% vol./vol. was prepared. and polysorbate 80 at 0.1% in pH 7.4 phosphate-buffered saline (PBS). A volume of 600 µl of the aqueous solution was added to the Kolliphor EL and drug during mixing and vortexed immediately. The pH was adjusted to 7.4 using 1 M NaOH in the formulation with stirring. The volume was adjusted to the target volume using an aqueous solution. The formulation was filtered using a 0.2 µm filter into a sterile vial inside a Class II biological hood.

Compound IIc-007a solution: A total of 50 mg of Kolliphor EL was transferred into a clean dry container. The exact amount of compound (target concentration 5 mg/ml) was measured and gradually added to Kolliphor EL with stirring on a magnetic stirrer. Heating to 30 °C helped to incorporate the drug into the Kolliphor preparation. In a separate container, an aqueous solution of HPMC at 0.5% vol./vol., povidone at 0.5% vol./vol. was prepared. and polysorbate 80 at 0.1% in pH 7.4 phosphate-buffered saline (PBS). A volume of 600 µl of the aqueous solution was added to the Kolliphor EL and drug during mixing and vortexed immediately. The pH was adjusted to 7.4 using 0.5 M NaOH in the formulation with stirring. The volume was adjusted to the target volume using an aqueous solution. The formulation was filtered using a 0.2 µm filter into a sterile vial inside a Class II biological hood. Formulations are ready for dosing. Before use, vortex compound IIc-007a solution for 2 minutes and use within 10 minutes.

* the appearance of a gel/colloidal structure can be observed, but it redisperses after vortexing.

Suspension of compound Ic-007a: A total of 50 mg of Kolliphor EL was transferred into a clean dry container. The exact amount of compound (target concentration 5 mg/ml) was measured and gradually added to Kolliphor EL with stirring on a magnetic stirrer. Heating to 30 °C helped to incorporate the drug into the Kolliphor preparation. In a separate container, an aqueous solution of HPMC at 0.5% vol./vol., povidone at 0.5% vol./vol. was prepared. and polysorbate 80 at 0.1% in pH 7.4 phosphate-buffered saline (PBS).

The aqueous solution was filtered using a 0.22 µm filter. A volume of 600 µl of the aqueous solution was added to the Kolliphor EL and drug during mixing and vortexed immediately. The pH was adjusted to 7.4 using 0.5 M NaOH in the formulation with stirring. The volume was adjusted to the target volume using an aqueous solution.

Prior to use, the suspension of compound Ic-007a was sonicated to break up flocculates/aggregates using the following protocol: a small petri dish/beaker was filled with water inside a sonicator. The vial was placed in a beaker/petri dish and sonicated for 10 minutes at 40 °C. After 10 minutes, the vial was removed and mixed for 2 minutes and sonicated again for 10 minutes. The process was repeated for a total of 50 minutes.

Ophthalmic formulations IIc-007a

Vehicle composition and compound concentration are shown in Table 29.

Table 29: composition of formulations A, B and C

To prepare a 10 mg/g solution, 10 mg of compound was accurately weighed into a vial and 1 ml of vehicle was added directly to the API and vortexed to obtain a solution.

To prepare a 5 mg/g solution, 5 mg of compound was accurately weighed into a vial and 1 ml of vehicle was added directly to the API and vortexed to obtain a solution.

Vehicle preparation

Formulation A: 50 g of vehicle was prepared by weighing 0.25 g of HPMC (0.5% vol./vol.) into a 100 ml duran glass bottle. 49.25 g of Type 1 water was then added to the bottle, followed by 0.200 g of TRIS (0.4% vol./vol.), 50 mg of Tween 80 (0.1% vol./vol.) and 0.25 g of PVP. K30 (0.5% vol./vol.). The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Formulation B: 50 g of vehicle was prepared by weighing 2.5 g of cremophor EL (5% vol./vol.) inside a 100 ml duran glass bottle. Next, 47.3 g of Type 1 water was added to the flask, followed by 200 mg of TRIS (0.4% vol./vol.).

Formulation C: 50 g of vehicle was prepared by weighing 2.5 g of cremophor EL (5% vol./vol.) inside a 100 ml duran glass bottle. Next, 46.45 g of Type 1 water was added to the flask, followed by 450 mg of TRIS (0.9% vol./vol.) and 600 mg of PVP K90 (1.2% vol./vol.). The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Formulation D: Diethylamine 0.3% w/v: 9.9 g of diethylamine vehicle was prepared by adding 9869 µl of Type 1 water to a 14 ml glass vial. Then, 44.2 µl (31.3 mg) of diethylamine was added to the vial using a pipette. The solution was left for 5 minutes with stirring to ensure homogeneity.

Ophthalmic formulations IVc-059a

Vehicle composition and compound concentration are shown in Table 30.

Table 30: compositions of formulations A, B and C

To prepare a 10 mg/g solution, 10 mg of compound was accurately weighed into a vial and 1 ml of vehicle was added directly to the API and vortexed to obtain a solution.

Vehicle preparation

Formulation A: 50 g of vehicle was prepared by weighing 2.5 g of cremophor EL (5% vol./vol.) into a 100 ml duran glass bottle. Next, 46.45 g of Type 1 water was added to the flask, followed by 450 mg of TRIS (0.9% vol./vol.) and 600 mg of PVP K90 (1.2% vol./vol.). The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Formulation B: Diethylamine 0.3% vol./vol.: 9.9 g of diethylamine vehicle was prepared by adding 9869 µl of Type 1 water to a 14 ml glass vial. Then, 44.2 µl (31.3 mg) of diethylamine was added to the vial using a pipette. The solution was left for 5 minutes with stirring to ensure homogeneity.

Formulation C: 50 g of vehicle was prepared by weighing 49.45 g of PBS (preparation method below) into a 100 ml Duran glass bottle. Successively, 250 mg HPMC (0.5% vol./vol.), 250 mg PVP K30 (0.5% vol./vol.) and 50 µl (liquid displacement pipette) tween 80 (0.1% vol./vol.) were added to a glass bottle. The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

PBS preparation: 799.1 mg NaCl, 21.6 mg KCl, 24.9 mg KH2PO4, 180.4 mg Na2HPO4·2H2O were accurately weighed and dissolved in 100 ml of type 1 water. The pH of the solution was 7.6.

Ophthalmic formulations Ic-007a and IIc-007a

The compounds are formulated as solutions in the vehicles shown in Table 31.

Table 31: compositions of formulations A, B, C and D

Preparation of the vehicle on a scale of 50 g

Vehicle A: 50 g of vehicle was prepared by adding 0.45 g of TRIS (0.9% v/v) and 2.5 g of Cremphor EL (5.0% v/v) to 47.05 g of water (94.1% v/v). The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Vehicle B: 50 g of vehicle was prepared by adding 0.45 g TRIS (0.9% vol./vol.), 2.5 g Cremphor EL (5.0% vol./vol.) followed by 0.1 g Kolliphor RH40 (0.2% vol./vol.) in 46.95% water (93.9% vol./vol.). The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Vehicle C: 50 g of vehicle was prepared by adding 0.45 g TRIS (0.9% vol./vol.), 2.5 g Cremphor EL (5.0% vol./vol.), followed by 0.1 g Kolliphor RH40 (0.2% vol./vol.) and 0.2 g PEG400 in 46.75 g water (93.5% vol./vol.). The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

Vehicle D: 50 g of vehicle was prepared by adding 0.45 g TRIS (0.9% vol./vol.), 0.5 g meglume (1.0% vol./vol.), 2.5 g Cremphor EL (5.0% vol./vol.) followed by 0.1 g Kolliphor RH40 (0.2% vol./vol.) to 46.45 g water (92.9% vol./vol.). The mixture was allowed to stir for 1 h at room temperature to allow all components to dissolve.

The vehicle was added directly to the API to create the concentrations listed in Table 32. The sample was vortexed and sonicated until a solution was obtained.

Table 32: Concentration of compounds in formulations using carrier A, B, C and D

To prepare a 10 mg/g solution, 10 mg of compound was accurately weighed into a vial and 1 ml of vehicle was added directly to the API and vortexed to obtain a solution.

To prepare a 20 mg/g solution, 20 mg of compound was accurately weighed into a vial and 1 ml of vehicle was added directly to the API and vortexed to obtain a solution.

EXAMPLE 5: Therapeutic activity of the compound

Example 5.1: Treatment and/or prevention of acute pancreatitis

The therapeutic efficacy of the compound according to the present invention was evaluated in a murine model of acute pancreatitis. Tissue samples were taken to allow for subsequent analysis of lifetime pancreatitis parameters. Plasma samples were also obtained to assess animal exposure levels following compound administration.

Animals: A total of 70 female Balb/c mice, 8-10 weeks old, weighing approximately 20-25 g, were used for the study (Charles River). After 7 days of acclimatization, they were assigned to different groups. Mice were housed in IVC cages (up to 5 mice per cage) with individual mice identified by tail.

Cages, bedding and water were disinfected before use. Animals were provided with corn cob enrichment bedding to provide environmental enrichment and nesting material. All animals had free access to a standard certified commercial diet and water. The room for keeping the animals was maintained as follows - temperature 20-24 °C, humidity 30-70% and a light/dark cycle of 12 hours was used. Although the animals used in this study were immunocompetent, preparation of dosing solutions and dosing/weighing of animals were performed in a sterile biological cabinet.

Test substance and formulation: in the first experiment, compounds Ia-001a, Ia-001aTz, Ic-001aTz/004a, Ic-007a, Ib-010a, IIIc-061a, IVc-059a were measured and dissolved in DMSO. The final formulation was in 5% DMSO, 10% Solutol, 10% PEG400, 75% 0.9% saline and the pH was adjusted to 7 with 0.5M NaOH solution.

In another experiment, compounds Ic-001aTz2, IIc-007a, IIIa-001aTz, IIIc-061a, IIIc-061a-E3, IVc-059a were measured and dissolved in DMSO. The final formulation was in 5% DMSO, 10% Solutol, 10% PEG400, 75% 0.9% saline and the pH was adjusted to 7 with 0.5M NaOH solution. For oral (PO) dosing, IVc-059a is formulated in water. Drug solutions were formulated on the day of dosing.

The purpose of this study was to evaluate the efficacy of the compound in a model of acute pancreatitis (14 days), generate tissue samples to allow post-life analysis of pancreatitis parameters and generate four plasma samples to assess animal exposure levels after the first and last injection obtained from blood samples taken 1h and 24h after the first and last injection.

Study design: On the first day of the study, animals were randomly assigned to treatment groups, ensuring an equal distribution of body weight. Mice (except for the negative control group) were treated with test compounds given 5 minutes prior to cerulein dosing, followed by 2 µg/kg intraperitoneal (IP) injection of cerulein daily throughout the study.

Table 33: First experiment.

Table 34: Second experiment.

Test compound treatment was given 5 minutes prior to cerulein dosing.

Table 35: Study schedule.

Serial observations

Body weight: body weight of all mice in the study was measured and recorded daily; this information was used to calculate the precise dosage for each animal.

General Signs and Symptoms: Mice were observed daily for any signs of distress or changes in general condition, eg, mottled fur, lack of movement, difficulty breathing.

Post-life sampling and analysis: At the above time points, a whole blood sample (60 µl) was collected from the lateral tail vein into K2-EDTA coated tubes. Plasma samples were prepared and stored frozen at -20 °C. Plasma samples were sent to the bioassay client Eurofins (FR) for compound quantification.

Terminal sampling: Before termination, the animals were weighed. Final study animals were sacrificed by CO2 inhalation. A terminal blood sample was taken by cardiac puncture and plasma was prepared. Lipase and amylase activities were measured by ELISA using commercially available ELISA kits. The remaining plasma was stored for future cytokine analyses. Pancreatic tissue was resected and weighed, and a section (50 µg) was frozen and analyzed for quantification of compounds in the pancreatic sample. A section (50 µg) was processed to measure MPO activity, IL-33 and TGF-B levels by ELISA. The remainder was fixed in formalin and embedded in paraffin wax. H&E staining was performed and the section was graded using the Schmidt standard scoring system, examining: edema, inflammatory infiltration, parenchymal necrosis, hemorrhage. Masson's trichrome staining was performed and the area covered by fibrous tissue was quantified digitally using QuPath software. Terminal serum samples were used for blood chemistry assessment using an IdeXX CHEM15 and LYTE4 clip (4 animals per treatment group).

Results of acute pancreatitis after 15 days of IV treatment

Histopathological assessment of pancreatic injury based on 4 criteria: Edema, inflammatory infiltration, parenchymal necrosis, hemorrhage, and score as described below.

Table 36: Score levels based on 4 criteria

Table 37: Score levels for animal groups

Schmidt score

The Schmidt score is an assessment of compound efficacy based on four histopathological criteria as described in the table below. Order of compounds with increasing Schmidt score (highest score = 10 is carulein induced by vehicle treatment and lowest score = 0 is non-challenged animals).

Schmidt score plot for the compounds in Figure 2 where all products show an improved Schmidt score compared to cerulein control animals.

Table 38: Table of Schmidt results and four parameters

As shown in the tables below, all compounds Ia-001a, Ia-001aTz, Ic-001aTz/004a, Ic-007a, Ib-010a, IIIc-061a, IVc-059a showed a significant decrease in IL33 levels in pancreatic tissue compared to the "induced" group. TGF-B levels were significantly reduced by Ic-007a, Ib-010a, IIIc-061a, IVc-059a. Global Schmidt scores were notably improved for all compounds, but especially for compounds IIIc-061a, IVc-059a.

Tables 39 and 40 below show pancreatic terminal MPO concentrations in a pancreatic tissue sample, IL33 tissue concentrations, and TGF-B concentrations.

Table 39:

Table 40:

In another experiment compounds Ic-001a-TZ(2), IIc-007a, Ia-015a, IIIc-061a, IVc-059a were evaluated at 15 mg/k in the same model using pirfenidone 100 mg/kg as a positive control.

Table 41: Results

Amylase: All compounds tested, except Ia-015a, resulted in significantly lower levels of amylase activity than in induced vehicle-treated controls, however, levels did not reach those of non-induced animals.

Lipase: Serum lipase activity was higher in induced and vehicle-treated animals than in non-induced animals. Treatment with the positive control pirfenidone, Ia-015a, IIIc-061a, and IVc 059a resulted in significantly lower lipase activity than vehicle-treated animals.

Myeloperoxidase (MPO): MPO activity in pancreatic tissue of induced and vehicle-treated animals was significantly higher than that of non-induced animals, indicating pancreatitis. Treatment with the positive control pirfenidone, IIIc-061a, and IVc-059a resulted in MPO activity levels that were significantly lower than the induced vehicle-treated controls.

IL-33 and TGF-B: All treatments, except IIIc-061a and IVc-059a dosed PO, resulted in pancreatic IL-33 levels that were lower than induced vehicle-treated controls. As with serum levels of TGF-B it resulted in significantly lower levels of TGF-B compared to induced vehicle-treated controls.

Clinically used Schmidt scoring criteria based on interstitial edema, leukocyte infiltration, acinar cell necrosis, and hemorrhage were used to evaluate H&E-stained pancreatic tissue sections. All compounds tested resulted in significantly lower Schmidt scores than induced vehicle-treated controls, with IIIc-061a (IV) and IVc-059a (IV) having lower scores than the pirfenidone positive control.

Fibrosis staining: A commercially available staining kit (Abeam; ab150686, Trichrome Stain) was used to stain pancreatic tissue sections for collagen. QuPath digital imaging software was used to quantify the percentage area covered by collagen (blue) staining. All tested compounds resulted in significantly lower areas of trichrome staining compared to induced vehicle-treated controls.

Example 5.2: Treatment and/or prevention of pancreatic and kidney cancer:

The aim of this study was to preclinically evaluate the in vivo study of the therapeutic efficacy of the compounds according to the subject invention for the treatment of a syngeneic model of pancreatic cancer in mice (Pan02) in female C57BL/6 mice and a syngeneic model of kidney cancer (Renca) in female BALB/c mice.

Animals: C57BL/6 female mice (for the Pan02 model); BALB/c female mice (for Renca model), 6-8 weeks, >17 g, up to 5 mice per cage

Pan02 model treatment and groups: compounds Ia-001a-Tz, Ic-007a, Ib-010a, IVc-059a, IIc-007a daily 5 days a week for two weeks via IV bolus injection at a dose of 15 mg/kg.

Renca model treatment and groups: compounds Ia-001a-Tz, Ic-007a, Ib-010a, IVc-059a, IIc-007a daily 7 days/week for three weeks via IV bolus injection at a dose of 15 mg/kg

Cell culture: Renca tumor cells were maintained in vitro with DMEM medium supplemented with 10% fetal bovine serum at 37 °C in an atmosphere of 5% CO2 in air. Cells in the exponential growth phase were harvested and quantified using a cell counter before tumor inoculation.

Cell culture: Pan02 tumor cells were maintained in vitro with RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C in an atmosphere of 5% CO2 in air. Cells in the exponential growth phase were harvested and quantified using a cell counter before tumor inoculation.

Tumor inoculation for the Renca model: each mouse was inoculated subcutaneously in the right hind flank region with Renca tumor cells (1 × 10<6>) in 0.1 ml of PBS for tumor development.

Tumor inoculation for the Pan02 model: each mouse was inoculated subcutaneously in the anterior right flank region with Pan02 tumor cells (3 × 10<6>) in 0.1 ml of PBS for tumor development.

Randomization: Randomization was initiated when mean tumor size reached approximately 100 (80 - 110) mm<3>. For both models, 48 mice were included in the study. All animals were randomly assigned to 6 study groups. Randomization was performed based on the "Paired Distribution" method using the multitasking method (StudyDirectorTM software, version 3.1.399.19). The date of randomization is designated as day 0.

Observation and data collection: After tumor cell inoculation, animals were checked daily for morbidity and mortality. During routine follow-up, animals were checked for any effects of tumor growth and treatment on behavior such as locomotion, food and water consumption, body weight gain/loss (body weight was measured twice a week after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs for individual animals were recorded in detail.

Tumor volumes were measured twice a week after randomization in two dimensions using calipers, and volume was expressed in mm3 using the formula: "V = (L × W × W)/2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). Tumor weight was measured at the end of the study. Dosing, as well as tumor and body weight measurements, were performed in the laminar flow.

Study endpoints: Tumor growth inhibition (TGI): TGI% was used as an indication of antitumor activity and was expressed as: TGI (%) = 100*(1-T/C) T and C were the mean tumor volume (or weight) of the treated and control groups, respectively, on a given day.

Statistical analysis of the difference in mean tumor volume between groups was performed using one of the following methods:

Use data collected on the last day of dosing/observation for each individual group, despite different individual cutoff dates.

Use data collected on the day when the mean tumor volume of the vehicle group reaches the human endpoints so that TGI can be performed for all/most mice included in the study.

Use data collected on the day that any of the treatment or control groups are terminated even if the remaining groups are treated as scheduled.

Difference in AUC (ΔAUC) = Statistical analysis performed with a mixed effects linear regression model. End of study: The efficacy study was conducted for 3 weeks.

Statistical analysis: To compare the tumor volumes of different groups on a predetermined day, Bartlett's test was first used to check the assumption of homogeneity of variance in all groups. When the Bartlett test p-value was ≥0.05, one-way ANOVA was used to test the overall equality of means across groups.

If the p-value of the one-way ANOVA was <0.05, post hoc testing was performed by running Tukey HSD (honest significant difference) tests for all pairwise comparisons and Dunnett tests to compare each treatment group with the vehicle group. When the p-value of the Bartlett test was <0.05, the Kruskal-Wallis test was used to test the overall equality of medians among all groups. If the Kruskal-Wallis p-value was <0.05, post hoc testing was performed by running the Conover non-parametric test for all pairwise comparisons or for comparisons of each treatment group with the vehicle group, both with a one-step p-value adjustment. All statistical analyzes were performed in the R-a language and environment for statistical computing and graphics (version 3.3.1). All tests are two-tailed unless otherwise noted, and p-values <0.05 were considered statistically significant.

Results

Table 42: Antitumor activity of test articles with data collected on day 13

Table 43: Statistical analysis of tumor volume with data collected on day 13

Summary of results

In this study, the in vivo therapeutic efficacy of test compound IVc-059a was evaluated for the treatment of a subcutaneous murine syngeneic model of pancreatic carcinoma (Pan02) in female C57BL/6 mice. Compound IVc-059a, administered at a dose of 15 mg/kg IV, significantly suppressed tumor growth, with a TGI value of 20.98% (P<0.05).

Table 44: Mean % inhibition of tumor volume

Pan02 model

Table 45: Mean % inhibition of tumor volume Renza model

Example 5.3: Treatment and/or prevention of diabetic retinopathy (DR)

Animal model: The db/db mouse model was used and compared with db/+ mice as a non-diabetic control. The db/db mouse carried a leptin receptor gene mutation and was a model for obesity-induced type 2 diabetes. Bogdanov et al. PLoS On, 2014, 9, e97302 reported that db/db mice reproduced features of the neurodegenerative process that occurred in the human diabetic eye. In addition, this model developed early diabetes-induced microvascular abnormalities (vascular leakage) (Hernandez et al. Diabetes 2016, 65, 172-187; Hernandez et al. Diabetologia 2017, 60, 2285-2298). Therefore, this model is considered a suitable model for testing the efficacy of the compounds of the present invention for the treatment of early stages of diabetic retinopathy (DR).

The effect of eye drops containing Ia-007a on diabetes-induced vascular leakage was tested in the db/db mouse model. Mice, 8-week-old db/db (BKS.Cg-Dock7m /+ Leprdb/J) mice and nondiabetic (db/+) mice were purchased from Charles River Laboratories (Calco, Italy).

Animals had free access to food ad libitum (ENVIGO Global Diet Complete Feed for Rodents, Mucedola, Milan, Italy) and filtered water. Mice were maintained under strict environmental conditions of temperature (20 °C) and humidity (60%). Moreover, they had 12 h/12 h light/dark cycles.

Intervention study: When db/db mice were 10 weeks old, Ia-007a eye drops or vehicle eye drops were randomly administered directly to the upper corneal surface of each eye using a syringe. One drop (5 µl) of Ia-007a (5 mg/ml) in each eye or vehicle (5 µl of 0.9% sodium chloride) in each eye was administered twice daily for 14 days. On day fifteen, animals were instilled with a drop of Ia-007a or vehicle approximately one hour before necropsy. All experiments were performed in accordance with the principles of the European Community (86/609/CEE).

Retinal vasculature permeability was examined ex vivo by assessing albumin leakage from retinal blood vessels using the Evans Blue albumin method. For this purpose, four animals per group were injected intraperitoneally with a solution of Evans Blue (E2129 SIGMA, Saint Louis, Missouri, USA) (5 mg/ml dissolved in PBS pH 7.4). After the injection, the animals turned blue, confirming uptake and distribution of the dye. After 2 hours, the mice were euthanized by cervical dislocation, and the eyes were enucleated. The retinas of each animal were isolated, measured, and rapidly protected from light. Flat slides were obtained and the cover slip was slid off a drop of Prolong Gold antifade reagent mounting medium (Invitrogen, Thermo Fisher Scientific, Oregon, USA). Digital images from different random fields of all retinas were acquired using a confocal laser scanning microscope (FV1000; Olympus, Hamburg, Germany) at ×60 using a 561 nm laser line, and each image was captured with identical beam intensity at a size of 1024 pixels × 1024 pixels. For quantitative analysis of albumin-bound Evans Blue, the number of extravasations per 0.44 mm<2> field was counted. This analysis was performed by investigators who were unaware of the treatment the mice received.

Results

Blood glucose concentration and body weight at the end of treatment were similar in db/db mice treated with Ia-007a eye drops than in vehicle-treated db/db mice. Extravascular locations of Evans Blue were identified in the retina of diabetic mice (Figure 1, white arrows). Topical treatment with Ia-007a was able to significantly reduce the number of extravasations per field, thereby preventing vascular leakage due to disruption of the retinal blood barrier.

Example 5.4: Treatment and/or prevention of peritonitis

The therapeutic efficacy of the compounds of the present invention was evaluated using the thioglycollate-induced peritonitis model in mice as described by Cook AD et al. (J Immunol. 2003;171(9):4816-4823) and Tsai JM et al. (Blood Adv.2019;3(18):2713-2721. doi:10.1182/bloodadvances.2018024026).

C57BL/6J mice were injected intraperitoneally (ip) with 1 ml of compound at a dose of 50 mg/kg 1 hour before the induction of peritonitis. Peritonitis was then induced with 1 ml of sterile 4% (wt/vol) thioglycollate broth or PBS as a control. After 3 h, mice were anesthetized by ip injection with a PBS solution containing 5 mg/ml ketamine and 1 mg/ml xylazine, and the peritoneal cavities were flushed with 5 ml ice-cold sterile Ca2+/Mg2+ HBSS 1X containing EDTA 2 mm. Peritoneal exudate cells (PEC) were centrifuged at 1200 rpm for 5 min at 4 °C and red cells were lysed. PECs were washed, resuspended in PBS 10% FBS, incubated with anti-CD 16/CD32 and mIgG FcR blockers and then stained with fluorescent conjugated antibodies to characterize migrated leukocytes (anti-CD45, anti-CD11b, anti-Ly6G, anti-CD8a, anti-CD4, anti-CD3). Cell viability was measured by 7-AAD exclusion. Samples were taken by flow cytometry using MACSQuant Analyzer (Miltenyi Biotec) and analyzed using FlowJo software. Animals: four C57BL/6J mice per group for each compound versus four vehicle-treated mice.

Treatment with cpds: 50 mg/kg ip compound injection

− Ic-007a: Volume injected: Ic-007a 68.5 µl compound in DMSO (1.375 mg) 431.5 µl PBS (500 µl total volume, 5.87 mm mouse about 28 g

− IIc-007a: Injected volume: 67.25 µl compound in DMSO (1.345 mg) 431.5 µl PBS (500 µl total volume, 5.74 mm) approx.27 g

− IIIc-061a: Injected volume: 58.75 µl compound in DMSO (1.175 mg) 441.25 µl PBS (500 µl total volume, 4.56 mm) approx.23.5 g − IVc-059a: Injected volume: 52.25 µl compound in DMSO (1.045 mg) 447.75 µl PBS (500 µl total volume, 5.25 mm) approx. 20.9 g

Treatment with products Ic-007a, IIc-007a, IIIc-061a, IVc-059a was performed 60 min before thioglycolate-induced peritonitis. The reading was taken 1 h after the injection of thioglycolate.

Reading method: flow cytometry for the characterization of migrated leukocytes using anti-CD45, anti-CD11b, anti-Li6G, anti-CD8a, anti-CD4, anti-CD3.

Results and statistical analysis: Results were analyzed using FlowJo software. Data are presented as mean ± SEM. A two-tailed Student t test using a significance level of 0.05 is used for statistical analysis.

Results

Table 46: Example data for compound Ic-007a

Table 47: summary of results for four compounds

Example 5.5: Treatment and/or prevention of diabetic cardiomyopathy

The therapeutic efficacy of the compounds of the present invention was tested using mouse models of diabetic cardiomyopathy described by Li C et al. (Cardiovasc Diabetol. 2019;18(1):15. Published February 2, 2019. doi:10.1186/s12933-019-0816-2).

Animal procedure and drug treatment: Thirty 8-week-old KK-Ay mice (a genetic model of type 2 diabetes) and C57BL/6J mice were housed in cages (4-6 per cage) with free access to drink/food boxes. Mice were housed in a room at 24 °C with a light/dark cycle of 12:12 h.

Mice to model type 2 diabetes are fed a high-fat diet. Blood glucose is measured daily. Mice with blood glucose concentrations greater than 15 mm (200 mg/dl) for 2 consecutive weeks were used for the following experiments. After that, the animals are randomized into groups (15 per group) and bred for 8 weeks. Groups include control groups: (1) healthy C57BL/6J mice without treatment; (2) KK-Ay mice with type 2 diabetes without treatment; and (3) type 2 diabetic KK-Ay mice treated with compounds of the present invention.

Echocardiographic evaluation: Echocardiographic measurement is performed before the experimental intervention and at the end of the research period. M-mode echocardiography equipped with a 17.5 MHz linear array transducer system (Vevo 2100<™>High Resolution Imaging System; Visual Sonics). Heart rate (HR) and the following structural variables are assessed: left ventricular internal dimension in diastole (LVIDD), left ventricular internal dimension in systole (LVIDS), interventricular septal thickness in systole (IVSs) and diastole (IVSd), and LV posterior wall thickness in systole (LVPWs) and diastole (LVPWd). LV mass is calculated using the formula [(LVIDd LVPWd IVSd)<3>- (LVIDd)<3>× 1.04 × 0.8 0.6]. LV function is assessed by the following parameters including fractional shortening (FS), ejection fraction (EF) and E/A ratio. All measurements were performed by a single investigator who was blinded to the experimental groups.

Myocardial Hydroxyproline Concentration: Myocardial hydroxyproline concentration is measured in the left ventricle to estimate myocardial collagen content. Measurements are performed with a spectrophotometer using a commercial kit (BioVision, Mountain View, CA, USA) according to the manufacturer's protocols.

Detection of serum lipid, glucose, insulin and HbA1c levels: At the end of the study, mice were anesthetized by inhalation of 3% isopentane in air. Fasting blood samples are collected into commercial tubes containing lithium heparin as an anticoagulant through the sublingual vein of each animal and centrifuged for 6 minutes at 3000 rpm. Plasma is stored in a regular tube and stored at -20 °C until analysis.

Concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in plasma are determined by spectrophotometric methods. Blood glucose levels are measured using a glucometer (ACCU-CHEK, Roche, USA). After overnight fasting, fasting insulin and proinsulin levels were determined using mouse insulin and proinsulin enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Biotech, China). Blood is also retained for HbA1c measurement using an ion exchange chromatography-spectrophotometric kit (Biosistems, Spain).

Assessment of oxidative stress in heart tissue: Animals were euthanized and hearts were removed and retrogradely washed with Krebs-Ringer solution (115 mm NaCl, 5 mm KCl, 1.2 mm KH2PO4, 25 mm NaHCOs, 1.2 mm mgSO4, 1.25 mm CaCl2 and 11 mm glucose) at the end of the study. The temperature of the perfusion solution is maintained at 37 °C. Hearts were then weighed, and cardiac tissues were homogenized on ice in chilled phosphate-buffered saline (PBS) at pH 7.4, containing 1 mM EDTA. Homogenates are centrifuged in cold physiological solution for 10 min at 7000 rpm. Protein concentration in the supernatant is determined using a bovine serum albumin kit as a standard. Supernatants are used for analysis of lipid hydroperoxide levels and levels of glutathione peroxidase (GSH-Pk), superoxide dismutase (SOD) and malondialdehyde (MDA). Measurements are performed with a spectrophotometer using commercial kits (Solarbio, China) according to the manufacturer's protocols. Lipid hydroperoxide level is expressed as nmol/mg protein. GSH-Px, SOD, and MDA levels were expressed as µmol/mg protein, nmol/mg protein, and mmol/mg protein, respectively. The expression level of NOX4 is measured by Western blotting. Mouse monoclonal anti-Nox4 (1:1000, Abcam) and horseradish peroxidase-conjugated secondary antibody (CVBIO) are used. β-Actin (1:1000, Abeam) is used as a reference.

Histological and immunohistochemical analysis: Heart tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 48 h, dehydrated and embedded in paraffin, sectioned at 4 µm thickness and mounted on glass slides. Masson's trichrome staining is used to assess the extent of fibrosis in the heart muscle.

For antigen retrieval, deparaffinized slides are kept in a solution of 10 mm sodium citrate (pH 6.0) for 10 minutes at 100 °C. Sections are then incubated in 3% hydrogen peroxide to block endogenous peroxidase activity and incubated with primary antibody (mouse monoclonal antibody to TGF-β1, collagen I and collagen III; Abcam) for 1.5 h, followed by the appropriate secondary antibody for 2.5 h at room temperature. After that, the sections were washed in PBS three times and incubated in 0.02% diamino benzidine solution for 2-8 minutes. After counterstaining with hematoxylin, the slides are briefly washed, mounted with a slide and observed under a light microscope.

Analysis of Nrf2/ARE and TGF-β/SMAD pathway by western blotting:

The Western blotting protocol was described in our previous study. Briefly, myocardial tissue was lysed in ice-cold RIPA buffer (150 mM sodium chloride, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1.0% NP-40, 1 mM PMSF, and 50 mM Tris, pH 8.0) for total protein extraction. Total protein concentration was quantified using the BCA Protein Assay Kit (Medchem Express, USA). Equal amounts of protein are separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE). The protein is then transferred from the gel to a polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane is incubated overnight in primary antibody (Nrf2, HO-1, TGF-β1, p-Smad2, Smad2, p-Smad3, Smad3, Smad7, α-SMA 1:1000, Abcam). Bands are detected with a specific horseradish peroxidase-conjugated secondary antibody (CVBIO). β-actin (1:1000, Abeam) is used as a total cellular protein reference.

Example 5.6: Treatment and/or prevention of diabetes and healing of diabetic wounds

Mouse models of diabetes using streptozotocin (STZ-induced diabetes) were used to evaluate the efficacy of compounds of the present invention for healing diabetic wounds and to generate tissue samples to allow post-life analysis of skin tissue.

Animals: A total of 104 female CD-1 mice aged 5-7 weeks, weighing approximately 25-30 g, were implanted for the study. They were purchased from Charles River and acclimatized for 7 days upon arrival.

Animal housing: Mice were housed in IVC cages (up to 5 mice per cage) with individual mice identified by tail. Cages, bedding and water were disinfected before use. Animals were provided with corn cob enrichment bedding to provide environmental enrichment and nesting material. Each cage was clearly labeled with a card indicating the number of animals, sex, strain, birth sex, study number, and start and treatment dates. The cages were changed once a week with replacement of food and water as needed.

The room for keeping the animals was maintained as follows - temperature 20-24 °C, humidity 30-70% and a light/dark cycle of 12 hours was used.

Aseptic Technique and Dosing: Although the animals to be used in this study are immunocompetent, preparation of dosing solutions and dosing/weighing of animals were performed in a sterile biological cabinet. IV Dosing: Compounds are formulated in 5% DMSO: 10% Solutol: 10% PEG400: 75% PBS and pH adjusted to 7 with 0.5M NaOH solution. PO dosage: the formulation is in water.

Drug solutions are formulated on the day of dosing, all remaining at the end of dosing are stored at -20 °C.

Study design: Mice (except the negative control group) were injected with STZ (55 mg/kg; IP) daily for 5 consecutive days. Mice were fasted for 6 hours before injection. One week after the end of induction glucose levels were monitored and mice with levels less than 15 mml/l (280 mg/gl) were removed from the study because they did not develop the ideal severity of diabetes. Animals were anesthetized and a 6 mm full-thickness wound was made using a sterilized biopsy punch. Wounds were recorded (0 days). Animals were treated and imaged every 2 days until day 14 (when control wounds from non-diabetic mice were healed). Wound images were digitally assessed for wound area and percent closure versus time plotted graphically.

Table 48:

Treatment will continue for 2 weeks (14 days). Animal numbers in parentheses are dosed over 7 days and then sampled for PD analysis mid-study.

Table 49: Study schedule:

Serial observations

Body weight: body weight of all mice in the study was measured and recorded daily; this information is used to calculate the precise dosage for each animal.

General signs and symptoms: mice were observed daily for any signs of distress or change in general condition, e.g. Spotted fur, lack of movement, difficulty breathing were observed.

Mid-Study Sampling: After 7 days of dosing, 3 animals per group were killed and the wound tissue removed and divided into 2 parts (A) and (B):

(A) The first section is FFPE and is stained with the following: H&E, Masson's trichrome, CD31, TGF-β.

(B) The second part is homogenized and used for the following ELISA tests: Levels of SOD, GTPx, catalase, TNF-α, IL-6, TGF-β.

Termination: Final study animals were killed by CO2 inhalation. Before termination, the animals are weighed. Early termination: Early termination of an animal is performed if weight loss of ≥20% is measured and for any endangered animal showing critical signs and symptoms or inability to eat/drink.

Results:

Blood Glucose: Induction with STZ resulted in a significant increase in blood glucose compared to non-induced vehicle controls. Treatment with Ia-001a, IIIc-061a, and IVc-059a IV resulted in a significant reduction in blood glucose levels compared to vehicle-induced controls. Already after 7 days, all compounds showed a certain degree of accelerated wound healing in the STZ-induced diabetes model (Figure 10, 3 animals per group sampled after 7 days of dosing). Skin tissue was sampled.

Example 5.7: Treatment and/or prevention of diabetic foot ulcers

Compounds of the present invention were tested using a rat diabetic ulcer model as described by Zhang Y et al. (J Diabetes Res. 2016, 2016, 5782904. doi: 10.1155/2016/5782904).

Wound healing in skin and other mucosal tissues involves a complex series of events including coagulation, acute and chronic inflammation, reepithelialization, granulation tissue formation, wound contraction, and connective tissue remodeling. However, several genetic and acquired conditions, such as aging, malnutrition (eg, vitamin C and protein deficiency), infection, hypoxia, and genetic diseases such as Ehlers-Danlos syndrome, can impair this reparative process. Among these conditions, diabetes mellitus is the most common cause of damaged or unhealed wounds. As an example, the clinical importance of long-term hyperglycemia is highlighted by alarming data showing that 85% of non-healing diabetic foot ulcers eventually require amputation. The "pathway to a chronic wound," as noted by the authors of a recent study, focuses on prolonged or chronic inflammation characterized by macrophage activation (as well as neutrophil accumulation) leading to elevated levels of proinflammatory cytokines, reactive oxygen species, matrix metalloproteinases (MMPs), and other neutral proteinases (eg, elastase). This, along with a lack of endogenous proteinase inhibitors, all lead to "excessive matrix degradation, growth factor degradation, and impaired epithelialization."

In the first experiment, fifteen adult male Sprague-Dawley rats (body weight 300-325 g, Charles River Laboratories International, Inc., Wilmington, MA) were injected through the tail vein with either 10 mm citrate saline buffer pH 4.5 (nondiabetic controls, NDC) or the same solution containing streptozotocin (STZ; ENZO Life Sciences, Inc., Plymouth Meeting, PA; 70 mg/kg body weight) to challenge of type I diabetes. The rats were then assigned to the five experimental groups described below (n = 3 rats/group). All rats were given unlimited access to food and water. Within 48 hours, STZ-injected rats showed severely elevated levels of glucose in their urine. Three weeks after the induction of diabetes, the back skin of all rats was shaved and a series of six standard wounds per rat, each 6 mm in diameter, were made using a surgical trephine. The following five experimental groups were formed (in this initial experiment, treatment in all groups lasted seven days; the long-term study is described below in Experiment 3): non-diabetic control (NDC) rats treated with daily topical application of white petroleum jelly (“vehicle”); diabetic rats (D group) locally treated daily with vehicle only; diabetic rats treated with test compounds.

At the end of this time period, six circular wounds per rat are clinically evaluated by measuring the diameter of the wounds in millimeters with a caliper, blood samples are taken, the rats are sacrificed, and skin samples are dissected for histological/histochemical and biochemical evaluation as described below.

On the seventh day after the creation of standardized wounds, all animals were anesthetized, blood samples were taken for blood glucose (One Touch ultra Glucometer; Johnson & Johnson, New Brunswick, NJ) and HbA1c (Bayer A1CNow Selfcheck, Sunnyvale, CA) measurements, and, after the procedures below were completed, the rats were sacrificed by CO2 inhalation.

Images are taken for clinical measurements to assess wound closure (18 wounds per experimental group). The percentage reduction in wound area is calculated by measuring the diameter (in millimeters) of each wound before and after the treatment protocol.

On day 7, wound tissues were excised from two sites per rat and collected for biochemical analysis. Each tissue pool was homogenized, extracted at 4 °C with 5 M urea in 50 mM Tris-HCl buffer (pH 7.8) containing 0.2 M NaCl and 5 mM CaCl2 overnight, and then centrifuged for 1 h at 11,000 × g as described by us previously. Supernatants are dialyzed against Tris-HCl, NaCl and CaCl2 buffers, and proteinases are partially purified by adding ammonium sulfate to 60% saturation. Precipitated proteinases were analyzed by ELISA for the collagenases mmP-8 (Sigma-Aldrich Life Sciences Inc., St. Louis, MO) and mmP-13 (TSZ Scientific LLC, Framingham, MA).

Biopsies of each of the two wound sites, including surrounding unwounded tissue, were taken and fixed in 10% neutral buffered formalin for 24 hours, then transferred to 50% ethanol prior to freezing, alcohol dehydration, xylon clearing, paraffin embedding, and sectioning. Five-micron sections were stained with H&E and Masson's trichrome, and the distance between wound edges was measured histomorphometrically using a calibrated ocular micrometer and confirmed image analysis. The last two wounds per rat were dissected, hydrolyzed, and analyzed for hydroxyproline as described below.

At the end of the 14- and 30-day treatment protocols, physical measurements and histological and histochemical evaluations were the same as those described above for the 7-day experiment. In addition, for all three time periods, tissue samples from 6 mm biopsies were hydrolyzed twice in 2 N NaOH at 120 °C for 1 hour each time. Aliquots of 50 µl of skin tissue hydrolyzate are then analyzed for hydroxyproline, an amino acid found essentially only in collagen.

Example 5.8: Treatment and/or prevention of diabetic retinopathy and diabetic nephropathy

The therapeutic efficacy of the compounds of the present invention was evaluated using a diabetic mouse model using the DBA/2 mouse strain with STZ induction of diabetes and monitoring of diabetic retinopathy including kidney damage. Five weeks after induction of diabetes by low-dose STZ injection for 5 consecutive days, the urinary albumin:Cr ratio was 424-fold compared with 36.9 for age-matched controls.

To treat diabetic nephropathy, DBA/2 mice were induced with STZ (low dose for 5 consecutive days). One week after the end of induction, glucose levels were monitored and mice with levels less than 15 mml/l (280 mg/0.1 l) were removed from the study because they did not develop diabetes of sufficient severity to cause kidney injury. Glucose levels were monitored weekly and 5 weeks after STZ induction biochemical assessment of kidney damage was performed by measuring urinary albumin and creatinine levels and confirming that they were within the desired range. After the successful completion of this phase, the animals were assigned to treatment groups.

Treatment was carried out for four weeks (IV dosing) with weekly assessment of blood glucose and urine albumin/creatinine levels. At the end of the animal treatment period, the following samples were taken:

- Blood for measuring glucose and blood urea nitrogen (BUN)

- Blood to measure serum creatinine

- Urine for final albumin/creatinine ratio.

− The kidney was resected and FFPE: Picro-Sirius Red, Masson's trichrome and H&E staining was performed to identify areas of fibrosis along with evidence of mesangial sclerosis, arteriolar hyalinosis, thickening of the glomerular basement membrane (GBM)

To assess the compound's effect on diabetic retinopathy, the animals had weekly images taken via a fundus camera. Immediately before sacrifice, animals were injected with FITC-dextran via the tail vein. During sampling, eyes were sampled and fixed in formalin. Retinal flatmounts were prepared and examined fluorescently by observing the effects of compounds on vascularity and vessel leakage (indicated by high background staining). Animal groups (n=8 mice per group):

Table 50:

Treatment was continued for 4 weeks (28 days) with weekly blood glucose and urine albumin/creatinine measurements.

Sacrifice after the last compound (agent) dosing, organ and blood sampling.

Table 51: Study schedule

Serial observations

Body weight: The body weight of all mice in the study was measured and recorded daily; this information was used to calculate the precise dosage for each animal.

General signs and symptoms: Mice were observed daily for any signs of distress or changes in general condition, e.g. fur with stars, lack of movement, difficulty breathing.

Sampling and subsequent analyzes in life

• Immediately prior to terminal sampling, 4 animals per group were injected with FITC-dextran for retinal evaluation on a flat surface.

• Terminal sampling:

∘ Terminal blood samples were taken by cardiac puncture and plasma/serum was prepared from each animal.

▪ Blood glucose is measured

▪ Measured serum creatinine

▪ BUN measured

▪ Urine ALB/CREATININE

▪ ELISA for CRP, TNF-α, IL-6 and TGF-β

▪ The remaining plasma was stored for cytokine analysis

∘ Kidney tissue was resected and measured

▪ One kidney was snap-frozen for possible bioanalysis or other analysis.

▪ One kidney was fixed in formalin and embedded in paraffin wax. • H&E and Masson's trichrome staining screening for

o mesangial and glomerulosclerosis,

o arteriolar hyalinosis

about GBM thickening

∘ Eye tissue to be FFPE

▪ The other eye (4 per treatment group) was used for retinal flat-mount analysis to measure the vascular pattern

▪ Area covered by vessels

▪ Assessment of vascular leakage

Termination: Final study animals were sacrificed by CO2 inhalation. Before the break, the animals were weighed.

Results: Compounds Ia-001a, IIIc-061, and IVc-059a significantly reduced blood glucose levels in the DBA/2 mouse strain with STZ-induced diabetes as shown in Figure 9 (one and two weeks shown).

Example 5.9: Treatment and/or prevention of Behçet's disease (BD)

The therapeutic efficacy of the compounds of the present invention was tested using a mouse model of Behçet's disease as described by ZHENG et al. (Acta Derm Venereol 2015; 95: 952-958).

Behcet's disease is a chronic, recurrent, multisystemic, inflammatory disorder that mainly affects the oral and urogenital mucosa and the uveal tract. Although the etiology and pathogenesis of Behçet's disease are unknown, numerous etiologies have been proposed, including environmental, infectious, and immunological factors; an autoimmune basis, characterized by circulating immune complexes and complement activation, is gaining increasing acceptance. To test and understand the immunopathogenesis of Behçet's disease, animal models have been developed based on environmental pollutants, peptides derived from bacterial and human heat shock proteins, and virus injections. Using these animal models separately and/or simultaneously allows for more efficient research on Behçet's disease.

Recently, animal models have been developed to find effective and safe treatment options. Neutrophil activation is one aspect of the immunopathogenesis of BD. Neutrophils play a key role in innate immune responses. As typical lesions of BD such as pustular folliculitis, pathergy reactions and hypopyon have significant neutrophil infiltrations, neutrophil functions and activation status were investigated. There are conflicting reports of increased, normal, or decreased basal and fMLP-stimulated superoxide production, phagocytosis, chemotaxis, and neutrophil-endothelial adhesion in BD. In a putative HLA-transgenic mouse model of BD, the only abnormality observed was increased superoxide release in response to fMLP. High superoxide responses were also present in HLA-B51+ patients and healthy controls in the same study.

A mouse model similar to Behçet's disease was developed by inoculating HSV type 1 grown in Vero cells into 4–5 week old male Institute for Cancer Research (ICR) mice. Briefly, the earlobes of experimental mice are scratched with a needle and inoculated with the virus twice, 10 days apart. Infected mice are observed for 16 weeks after the final inoculation. HSV inoculated mice having at least 2 Behcet-like symptoms were used as BD mouse model (n=5). Both uninfected ICR mice (n = 2) and HSV inoculated, but asymptomatic, mice.

Example 5.10: Treatment and/or prevention of uveitis (UVE)

The therapeutic efficacy of the compounds of the present invention was tested using animal models of autoimmune uveitis including endotoxin-induced uveitis (EIU) in rats as described by Fruchon S et al. (Molecules. 2013;18(8):9305-9316. Published August 5, 2013. doi:

10.3390/molecules18089305). This model is considered a clinically relevant model for human anterior uveitis. It consists in the systemic application of lipopolysaccharide (LPS), which leads to an acute inflammatory response in the anterior and posterior segments of the eye with the breakdown of the blood-ocular barrier and infiltration of inflammatory cells. The clinical signs of EIU mirror the changes seen in the disease in humans. The therapeutic effect of the compounds of the present invention was therefore tested in a rat model of EIU, in comparison with the "gold standard" dexamethasone.

Animals: Only animals without visible signs of eye defects are included. Animals are examined during the pre-test period, with particular attention paid to the eyes. They are kept under observation for a week before the experiment. Animals were housed individually in standard cages and had free access to food and tap water.

Ocular tolerance study: The study consisted of three groups of three male Sprague-Dawley rats. On day 0, animals were weighed, anesthetized, and administered a single intravitreal injection of 5 µl in both eyes. The first group receives a physiological vehicle (NaCl 0.9%), and the other groups receive different doses of the compounds of the present invention. Each animal is then evaluated by clinical observation and eye examinations. Eye examinations include funduscopy, slit-lamp corneal examination (SLE) using fluorescein dye that allows for McDonald-Shadduck scoring. The McDonald-Shadduck scoring system deals with: conjunctival parameters (congestion, swelling and discharge), aqueous flame (intensity of the Tindall phenomenon) as presumed evidence of a breakdown of the blood-aqueous barrier; injection of secondary and tertiary vessels into the iris; turbidity, its relative surface area, neovascularization and epithelial integrity (labeling with fluorescein) of the cornea; lens integrity. After the final eye examination, all animals are sacrificed. Eyes were collected at autopsy, fixed in modified Davidson's solution for 12 h, then 10% neutral buffered formalin, and processed for histology. Hematoxylin-eosin-stained tissue sections are evaluated under a light microscope by a board-certified veterinary pathologist.

Endotoxin-induced uveitis (EIU) rat model: Thirty-six female albino Lewis rats were randomly divided into six groups of six animals each. EIU is induced by injection of 100 µl sterile pyrogen-free saline containing 200 µg LPS (lipopolysaccharide from Salmonella typhimurium, Sigma-Aldrich, Saint-Quentin, France). Animals were treated immediately before induction of EIU with an intravitreal injection of 5 µl in both eyes of saline (NaCl 0.9%) containing no active ingredient, or with the test compounds, or 20 µg of dexamethasone. Animals are examined using a slit lamp (SLE) at 24 h, ie, the clinical peak of disease in this model. The intensity of clinical eye inflammation is evaluated on a scale of 0 to 5 for each eye. A score of 0 indicates the absence of inflammation. Grade 1 indicates the presence of minimal vasodilation of the iris and conjunctiva, but without observation of foci or cells in the anterior chamber (AC). Grade 2 indicates the presence of moderate dilatation of the iris and conjunctival vessels, but no evident dilatation or cells in the AC. A score of 3 indicates the presence of intense dilatation of iris blood vessels, flare-ups, and less than ten cells per slit-lamp field in AC. Grade 4 indicates the presence of more severe clinical signs than grade 3, with more than ten cells per slit-lamp field in the AC, with or without hypopyon formation. Grade 5 indicates the presence of an intense inflammatory reaction, fibrin formation in AC and complete isolation of the pupil. At the end of the experiment, ie 24 h after LPS challenge, the rats were anesthetized with an intraperitoneal injection of pentobarbital (30 mg/kg) and then killed with a lethal dose of pentobarbital.

Measurement of cytokine concentrations in ocular fluids: Aqueous fluid and vitreous from both eyes of each animal were collected after sacrifice. Multiplex analysis determines the amounts of pro-inflammatory T helper cytokines TNFα, IL-1β, IL-2, IL-6, IL-17 and IFNγ, as well as anti-inflammatory cytokines IL-4 and IL-10.

Measurement of serum cytokine concentration: Sera from three groups of rats (saline vehicle, compound 10 µg and dexamethasone) were collected at the end of the experiment and stored at -80 °C. They are used for the simultaneous determination of five cytokine levels (IFNγ, TNFα, IL-2, IL-4 and IL-10) by Cytometric Bead Array (rat CBA Flek set, BD Biosciences, San Jose, CA, USA) on a FACS Calibur flow cytometer (BD Biosciences) according to the manufacturer's instructions. Amounts of each cytokine were analyzed against standard curves using FCAP Array software (BD Biosciences).

Example 5.11: Treatment and/or prevention of diabetic sensorimotor polyneuropathy and diabetic neuropathy

The therapeutic efficacy of the compounds of this invention was tested using rat models of diabetic peripheral neuropathy (Kambiz S. et al. (PLoS One.2015;10(6):e0131144]; PLoS One.2015;10(5):e0126892. Published May 18, 2015. doi:10.1371/journal.pone.0126892).

Animals: Female VAG/RijHsd rats (n = 27, 10 weeks old, weight 130-150 grams) were purchased from Charles River (l'Arbresle, France). Animals were housed in pairs in hooded cages at room temperature on a 12-h light/dark schedule and given water and food ad libitum.

Induction of diabetes: Diabetes was induced in 21 rats by a single intraperitoneal injection of STZ (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 65 mg/kg body weight in 0.05 mol/l sodium citrate buffer, pH 4.5, as previously described. Rats were randomly assigned to 3 groups: A, B and C (n = 7 in each group). After induction of diabetes, group A was killed after 4 weeks, group B after 6 weeks and group C after 8 weeks. The control group consists of 6 rats receiving a single intraperitoneal injection with an equal volume of vehicle without STZ. Control rats were followed for 8 weeks. Blood glucose was measured from tail vein blood using a glucometer (OneTouch, Life Scan, Milpitas, CA, USA). Diabetes is diagnosed in rats when the blood glucose level is greater than

20 mmol/l during the entire 4 weeks after the induction of diabetes.

Hindpaw blood flow and skin oxygenation: A combined laser Doppler flowmetry and spectrophotometry system (O2C, LEA Medizintechnik, Giessen, Germany) is used to noninvasively measure blood flow and oxygen saturation of bare hindpaw feet. In both diabetic and control rats, percent oxygen saturation and skin blood flow were assessed at 4, 6, and 8 weeks.

Heating rate after cold exposure: Skin temperature was assessed using a built-in infrared digital video camera (320 × 240 pixels) with a 1 Hz data acquisition system (ThermaCAM Researcher 2001 HS; FLIR Systems, Berchem, Belgium), and all data were continuously collected using a laptop. The distance between the camera and the hind paw is 13 cm ± 2 cm. The pixel size of temperature images is 0.8 × 0.8 mm. The skin temperature of the entire plantar hindpaw is recorded while the animal is fixed after the animal is placed on a 14°C plate for 5 seconds. Minimum hindpaw temperature was exported to text files using ThermaCAM Researcher Pro (version 2001-HS; FLIR Systems, Wilsonville, Oregon, USA). The area of interest is selected by drawing a line encircling the entire plantar hind paws. The average heating rate is shown as an increase in skin temperature in 120 seconds.

Thermal sensitivity: To determine the occurrence of thermal hypersensitivity, cold and hot plate tests are performed as previously described. Briefly, rats were placed in an open chamber with clean walls with a surface temperature of 5 °C (cold plate) or 50 °C (hot plate). These experiments are performed on separate days to prevent interference. The time until hindpaw withdrawal or licking is noted.

Von Frey test: In the Von Frey test, which is used to determine the mechanical sensitivity threshold for nociception, each rat is placed in a chamber with a mesh metal floor. Then, Von Freydlake, ranging from 2 to 300 grams, are applied 5 times, and the result is positive when at least 3 paw movements (reflex withdrawal response of the animal) are observed, as previously described. The control group served as the reference group.

Electromyography (EMG): Motor axon innervation in muscles is assessed by recording evoked CMAP peak-peak amplitudes and latencies of the gastrocnemius muscle in diabetic groups and control animals. CMAP peak-peak amplitude and latency are recorded and averaged for a group of 20 responses.

The average amplitudes in each diabetes group were compared with the control group. MNCV is calculated as the distance from the stimulating electrode to the recording electrode (mm)/latency (ms). Tissue preparation: After 4, 6, or 8 weeks, animals were killed by an overdose of pentobarbital (100 mg/kg ip). For each rat, the plantar skin of the hind paw was dissected and directly fixed by immersion in 2% paraformaldehyde-lysine-periodate (PLP) for 24 hours at 4 °C. The skin is further processed and embedded in gelatin as previously described. Finally, the embedded skin was sectioned at 40 µm with a freezing microtome and collected in glycerol for long-term storage at -20 °C.

Rat pancreatic tissue was harvested, fixed in 10% neutral buffered formalin, and embedded in paraffin. Afterwards, these samples were stained with hematoxylin and eosin (H&E). Each sample is evaluated using a bright-field microscope and scanned into digital slides (Nanozoomer 2.0 series, Hamamatzu, Japan).

Immunohistochemistry: Immunohistochemistry of skin sections was performed as previously described to semiquantify the density of sensory nerve fibers innervating the skin and to assess the presence of CD-31 positive endothelial cells. Skin sections were incubated for 48 hours in a cocktail of 2% BSA containing diluted primary antibody Protein Gene Product 9.5 (PGP9.5, 1/10,000, anti-rabbit, Life Sciences, New York, USA) or anti-CD31+ (1/5000, anti-rabbit, Spring Bioscience, California, USA) at 4 °C. Subsequently, the skin sections were incubated with the appropriate biotinylated horseradish peroxidase (HRP)-labeled secondary antibody (1/200, Biotine, Sigma-Aldrich, St. Louis, MO, USA) for 90 min at room temperature. The 3,-3' diaminobenzidine (DAB) reaction is then used to reveal the antigen binding sites of the primary antibodies. Afterwards, the sections were mounted on slides, and the CD31+ stained sections were counterstained with 0.05% thionin for 4 minutes, which stained the keratinocytes blue. Finally, all skin sections were dehydrated using absolute ethanol (< 0.01% methanol), transferred to xylene, and coverslipped with Permount (Fisher Scientific, Hampton, NH).

Each skin section was scanned in 3 slices of 8 µm each using a Nanozoomer 2.0 series (Nanozoomer 2.0 series, Hamamatzu, Japan). Four proximal and 4 distal sections of plantar hindpaw skin were quantified for epidermal nerve fibers in the central part of the plantar hindpaw (80,000 µm2) using a 40x objective in ImageScope software (Aperio ImageScope v11.12.760). Average labeled nerve fibers per mm2 and average epidermal thickness were calculated for each rat. The percentage of CD31-positive cells was calculated by Leica Cell-D (Olympus, Life Science Microscopy Imaging Software, USA) in 4 proximal and 4 distal skin sections over the entire upper dermis of the plantar hindpaw.

Example 5.12: Treatment and/or prevention of intestinal fibrosis

The therapeutic efficacy of the compounds of the present invention was tested using rat or mouse models as described by Pham BT et al. (Physiol Rep. 2015;3(4):e12323. doi: 10.14814/phi2.12323).

Preparation of rat and mouse intestinal cores: Non-fasted adult male Wistar rats and C57BL/6 mice (Harlan PBC, Zeist, The Netherlands) were used. Rats and mice were housed on a 12-h light/dark cycle in a temperature- and humidity-controlled room with food (Harlan chow no 2018, Horst, The Netherlands) and water ad libitum. The animals were allowed to acclimatize for at least seven days before the start of the experiment.

Rats and mice were anesthetized with isoflurane/O2 (Nicholas Piramal, London, UK). Rat jejunum (approximately 25 cm distal to the stomach and 15 cm in length) and mouse jejunum (approximately 15 cm distal to the stomach and 10 cm in length) were excised and stored in ice-cold Krebs-Henseleit buffer (KHB) supplemented with 25 mM d-glucose (Merck, Darmstadt, Germany), 25 mM NaHCO3 (Merck), 10 mM HEPES (MP Biomedicals, Aurora, OH), saturated with carbogen. (95% O2/5% CO2) and adjusted to pH 7.4. The jejunum is cleaned by flushing KHB through the lumen and then divided into 2 cm segments. These segments were loaded with a 3% (wt/vol) agarose solution in 0.9% NaCl at 37 °C and embedded in an agarose core embedding unit.

Preparation of human intestinal cores: Healthy human jejunal tissue is obtained for research from intestine resected from patients undergoing pancreaticoduodenectomy.

Healthy jejunum is stored in ice-cold KHB until the embedding procedure. The submucosa, muscularis, and serosa are carefully removed from the mucosa within one hour of tissue collection. The mucosa was divided into 0.4 cm × 1 cm sheets. These sheets were embedded in a 3% agarose solution (wt/vol) in 0.9% NaCl at 37 °C and inserted into the embedding unit.

PCIS preparation: PCIS is prepared in ice-cold KHB by Krumdieck tissue slicer (Alabama Research and Development). The 3-4 mg wet weight slices had an estimated thickness of 300-400 µm. Slices are stored in ice-cold KHB until the start of experiments.

Incubation of intestinal slices: Slices are incubated in 12-well plates for human PCIS (hPCIS) and rat PCIS (rPCIS) or in 24-well plates for mouse PCIS (mPCIS). hPCIS and rPCIS were incubated individually in 1.3 ml and mPCIS in 0.5 ml of Williams Medium E with L-glutamine (Invitrogen, Paisly, UK) supplemented with 25 mM glucose, 50 µg/ml gentamicin (Invitrogen) and 2.5 µg/ml amphocin-B (Invitrogen). During incubation (at 37 °C and 80% O2/5% CO2) in an incubator (MCO-18M, Sanio), the plates are shaken horizontally at 90 rpm (amplitude 2 cm). rPCIS were incubated for up to 24 h, mPCIS and hPCIS were incubated for up to 72 h, with and without human TGF-β1 (Roche Diagnostics, Mannheim, Germany) in the concentration range of 1 to 10 ng/ml. All incubations are performed in multiples (using 3-6 slices incubated individually in separate wells) and repeated with intestines from 3 to 16 different humans, rats, or mice.

Viability and morphology: Viability is assessed by measuring the content of adenosine triphosphate (ATP) in the PCIS. Briefly, after incubation, slices were transferred to 1 ml of sonication solution (containing 70% ethanol and 2 mm EDTA), snap-frozen in liquid nitrogen, and stored at -80 °C. To determine viability, ATP levels were measured in the supernatant of samples that had been sonicated for 45 seconds and centrifuged for 2 minutes at 4°C at 16,000 × g using an ATP bioluminescence kit (Roche Diagnostics, Mannheim, Germany). ATP values (pmol) were normalized to the total protein content (µg) of PCIS estimated by the Lovri method (BIO-rad RC DC Protein Assay, Bio Rad, Veenendaal, The Netherlands). To assess morphology, incubated slices were fixed in 4% formalin and embedded in paraffin. Sections of 4 µm were cut and stained with hematoxylin and eosin (HE). HE sections are scored according to the modified Park score, which describes the sequence of development of intestinal tissue injury after ischemia and reperfusion. The integrity of the seven segments of the PCIS is scored on a scale of 0 to 3. The viability of the epithelium, stroma, crypts, and muscle layer is scored separately with a score of 0 if there is no necrosis and 3 if massive necrosis is present. Other parts of the intestinal section were scored as follows: Epithelial shape: 0 = cuboidal epithelium, 3 = more than 2/3 of the cells are flat, flattened villi: 0 = normal, 3 = more than 2/3 of the villi are flattened, and the amount of edema: 0 = no edema, 3 = severe edema. A maximum score of 21 indicates severe impairment. In human samples, the morphological score of the muscularis mucosae is determined in the "muscular layer" section. B.T.P., W.T.v.H. and J.N. performed blind scoring; the mean value of the three total results is calculated.

Gene expression: Expression of fibrosis genes, namely COL1A1, αSMA, HSP47, CTGF, FN2, TGF-β1, PAI-1 and SIN was determined by either Taqman or SIBRgreen method. In hPCIS, ELA gene expression was also measured by the SIBRgreen method.

Intestinal fibrosis is a serious but common outcome in patients with inflammatory bowel disease (IBD). Despite progress in the development of new treatment modalities to control the chronic intestinal inflammation characteristic of IBD, to date there are no effective antifibrotic therapies. As such, a deeper understanding of the molecular mechanisms underlying intestinal fibrosis and the availability of relevant animal models are critical to moving this area of research forward.

IL-17 and associated mediators: Th17 cells define a subset of T helper cells that mainly produce IL-17A, but also IL-17F, IL-21, and IL-22, and are increasingly recognized as important in several chronic inflammatory disorders, including IBD (7). IL-17A has been implicated in fibrosis in multiple organs, including the lung (8), liver (9), and heart (10); recent studies also support its role in the intestine, linking IL-17/Th17 immune responses and other related mediators in the pathogenesis of intestinal fibrosis.

Other animal models of intestinal fibrosis are described below:

Table 52:

Example 5.13: Treatment and/or prevention of ovarian fibrosis

The therapeutic efficacy of the compounds of the present invention was tested using the ovarian hyperstimulation syndrome (OHSS) mouse model described by Pala et al. (Drug Des Devel Ther.2015;9: 1761-1766. Published March 24, 2015 doi: 10.2147/DDDT.S75266) to investigate the effects of test compounds on ovarian histopathology, serum VEGF and endothelin 1 levels in ovarian hyperstimulation syndrome (OHSS) in an experimental setting.

Animals: Female Wistar albino rats, 22 days old and randomly divided into groups, were used. Follicle-stimulating hormone 10 IU was administered subcutaneously to 15 rats for 4 consecutive days, with induction of OHSS on day 5 with 30 IU human chorionic gonadotropin. Group 1 includes 35-day-old control rats, group 2 includes 35-day-old OHSS rats, group 3 includes 27-day-old OHSS rats that received the test compounds for 7 days. All rats were then decapitated on day 35. Serum VEGF, endothelin 1 and ovarian follicular reserve were assessed in all rats.

Hematocrit and weight were measured on day 13, and decapitation was performed on day 35 under general anesthesia with intraperitoneal administration of ketamine (75 mg/kg) and xylazine (10 mg/kg).

Enzyme-linked immunosorbent assay: Approximately 3 ml of blood sample obtained from each decapitated rat. Sera were separated by centrifugation of blood samples at 2,500 rpm for 4 min and stored at -20 °C until assayed for VEGF and endothelin 1. VEGF was assayed using a mouse VEGF enzyme-linked immunosorbent assay kit (ELM-VEGF-001; RaiBio, USA) and endothelin was measured using endothelin 1 E/Kit (EK-023-01; Phoenix Pharmaceuticals, USA).

Ovarian morphology: After laparotomy, ovaries are removed and cleared of adherent tissue in culture medium and measured. Ovarian tissue was fixed with 10% formaldehyde, and then paraffin-embedded tissue samples were cut into 4 µm cross-sections for estimation of the mean number of ovarian follicles. Sections were stained with Masson's trichrome to determine OFR under light microscopy (Olympus BX-50). Transverse sections of 4 µm thickness were placed at 50 µm intervals on microscope slides to prevent counting the same structure twice, according to the previously described method. 12 follicles were classified as primordial, primary, secondary and tertiary. An atretic follicle (AF) is defined as a follicle presenting more than ten pyknotic nuclei; for the smallest follicles, the criteria for atresia are a degenerated oocyte, premature antrum formation, or both.

Main outcome measures: Main outcome measures are age (days), weight (g), hematocrit (%), ovarian weight (mg), serum VEGF (pg/ml) and endothelin 1 (ng/ml) levels, and total follicle count with determination of primordial, primary, secondary and tertiary follicle counts.

Example 5.14: Treatment and/or prevention of polycystic syndrome

ovaries (PCOS)

The therapeutic efficacy of the compounds of the present invention was tested using an animal model of DHEA-induced ovarian hyperfibrosis, as described by Wang D et al. (J Ovarian Res.2018;11(1):6. Published January 10, 2018. doi:10.1186/s13048-017-0375-7).

Polycystic ovary syndrome (PCOS) is a common metabolic and endocrine disorder with pathological mechanisms that remain unclear. The following study investigates ovarian hyperfibrosis formed through the transforming growth factor-β (TGF-β) signaling pathway in a dehydroepiandrosterone (DHEA)-induced rat model of polycystic ovary syndrome (PCOS).

Animals: Thirty female Sprague-Dawley (SD) rats, 21 days old, weighing ~50 g were used for this test. Animals were housed in a specific pathogen-free (SPF) environment with a temperature of 22 ± 1 °C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. Free access to food and water is provided.

Procedures: Thirty female Sprague-Dawley rats were randomly divided into a blank group (n = 6), an oil group (n = 6), and a DHEA oil-induced model group (n = 6 12). Model groups are established by subcutaneous injection of DHEA for 35 consecutive days. The rats were further divided into a vehicle group (n = 6) and a treatment group (n = 6). The treatment is given once a day and lasts 35 days. Eighteen days after treatment, vaginal smears were collected from all rats on a daily basis by assessing cell types for 14 days to determine their daily estrous cycles.36. day, all rats in the nothing and oil-treated groups and six rats in the DHEA-challenged model groups were euthanized (using an intraperitoneal injection of excess 5% chloral hydrate), blood was collected (from the superior vena cava), bilateral ovaries and uteri were dissected.

Ovaries were fixed in 4% paraformaldehyde for 24 hours at 4 °C and then embedded in paraffin. The rest of the tissue was frozen at -80 °C for further Western blotting and real-time polymerase chain reaction (RT-PCR) analysis.

The remaining 12 rats from the DHEA-induced model groups were randomized into two additional groups: a treatment group and a vehicle treatment group (control group), six rats per group. During this treatment, DHEA is no longer given to the rats. The treatment lasts 2 weeks, after which all animals are euthanized, blood is taken and both ovaries and uteri are dissected according to the above instructions.

Ovarian morphology, fibrin and collagen localization, and ovarian expression were detected using H&E staining, immunohistochemistry, and Sirius red staining. Ovary protein and RNA were examined using Western blot and RT-PCR.

Estrous cycle: On day 18 after DHEA treatment, vaginal smears were collected from all rats. The samples are then treated with toluidine blue for 30 minutes, and subsequently cell morphology and estrous cycles are examined under an optical microscope (Leica Microsystems, Germany).

Serum hormone levels: Blood samples are taken from the superior vena cava. Serum was immediately separated and stored at -20 °C for further determination of hormones by enzyme-linked immunosorbent assay (ELISA) (testosterone (T), estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH)) (rat T, E2, LH and FSH ELISA kits, USCN, Wuhan, China).

Example 5.15: Treatment and/or prevention of primary biliary cholangitis (PBC)

The therapeutic efficacy of the compounds of the present invention was evaluated using the PBC mouse model as described by Hohenester S et al. (Cells. 2020, 9(2), 281. doi: 10.3390/cells9020281).

Cholestatic liver diseases such as primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC) are chronic progressive disorders that often result in liver cirrhosis, with subsequent complications. Hydrophobic bile salts are thought to promote liver fibrosis in cholestasis. However, evidence for this widely accepted hypothesis remains scarce.

In established animal models of cholestasis, for example, Mdr2 knockout, cholestasis and fibrosis are secondary to biliary damage.

Therefore, to test the specific contribution of bile salt accumulation to liver fibrosis in cholestatic disease, a unique model of inducible hepatocellular cholestasis in cholate-fed Atp8b1G308V/G308V mice was used. Glycochenodeoxycholate (GCDCA) was supplemented to humanize the mouse bile salt pool as confirmed by HPLC. Biomarkers of cholestasis and liver fibrosis were quantified. Hepatic stellate cells (HSCs) isolated from wild-type mice were stimulated with bile salts. Proliferation, cell accumulation and collagen deposition of HSCs are determined. In cholestatic Atp8b1G308V/G308V mice, increased hepatic expression of αSMA and collagen1a mRNA and excess collagen deposition in the liver indicated the development of liver fibrosis only after GCDCA supplementation. In vitro, myofibroblast numbers and collagen deposition were increased after incubation with hydrophobic but not hydrophilic bile salts and associated with EGFR and MEK1/2 activation. Bile salts may have direct profibrotic effects on HSCs, reportedly involving EGFR and MEK1/2 signaling.

Animal experiments: Male animals are used for in vivo studies at the age of 8 weeks. Animals are maintained on a 12-hour light-dark cycle and housed in an enriched environment with ad libitum access to food and water.

Serum Biochemistry and Serum Bile Salt Measurements: Serum alkaline phosphatase, bilirubin, and alanine aminotransferase levels were quantified from fresh serum in a fully automated respons<®>910 analyzer (DiaSys, Holzheim, Germany). Serum total bile salt levels were quantified enzymatically using the Diazyme Total Bile Salt Kit (Diazyme Laboratories, Poway, CA, USA) according to the manufacturer's instructions.

Liver histology, immunohistochemistry and hydroxyproline quantification:

Paraffin blocks were cut into 4 µm thick slices and mounted on microscope slides (Superfrost plus, Thermo Scientific/Menzel, Braunschweig, Germany).

After gradual deparaffinization and rehydration, slides are stained with hematoxylin and eosin according to standard procedures. Immunohistochemistry was performed against alpha-SMA, using a monoclonal rabbit anti-alpha smooth muscle actin antibody (Abcam, Cambridge, UK). For collagen quantification, slides were stained for 1 h with Direct Red 80 (Sirius Red, Sigma-Aldrich, Darmstadt, Germany) and counterstained twice in ethanol and once in xylene. To quantify total DNA as a surrogate for cell number, HSCs are incubated with PicoGreen<®>(Invitrogen, Carlsbad, CA, USA) and fluorescent signals are detected using the CitoFluor 4000 system (PerSeptive Biosystems, Framingham, MA, USA). HSC proliferation is quantified using the BrdU assay kit (Roche, Penzberg, Germany) according to the manufacturer's instructions. To quantify the total number of cells, HSCs, seeded in Lab-Tek II Chamber Slides (Nunc, Rochester, NI, USA), were mounted on coverslips with Vectashield mounting medium including DAPI (Vector, Burlingame, CA, USA). Slides are scanned with a Pannoramic Midi Slide Scanner (3DHistech, Budapest, Hungary), and nuclei are counted with ImageJ2 software on the entire slide (0.7 cm2).

Quantification of collagen in vitro: Cells were washed with PBS and stained for 1 h with 0.1% Sirius Red in saturated picric acid. Cells were then washed three times with 100% ethanol, bound dye was dissolved in 50% methanol/sodium hydroxide (50 mmol/l), and absorbance was measured at 540 nm.

Example 5.16: Treatment and/or prevention of liver fibrosis or cirrhosis (HF)

The therapeutic efficacy of the compounds of the present invention to inhibit spontaneous, chronic inflammation and liver fibrosis was evaluated using established mouse models of NOD-inflammatory fibrosis (N-IF), as described by Fransen Pettersson et al. (PLoS One.2018 13(9): e0203228. doi:

10.1371/journal.pone.0203228).

Animals: N-IF mice spontaneously develop chronic inflammation and liver fibrosis induced by T-cell receptor (TCR) transgenic natural killer T (NKT)-II cells generated in immunodeficient NOD.Rag2-/- mice.

Several components of liver pathology in N-IF mice overlap with those of human conditions in which progressive chronic inflammation precedes the development of fibrosis.

Moreover, the fibrosis that develops in the liver of N-IF mice was shown to be associated with the accumulation of α-SMA-expressing hepatic stellate cells. These phenotypic similarities make the N-IF mouse model unique compared to other available rodent models of liver fibrosis and provide a new tool for testing the efficacy of anti-fibrosis drug candidates.

Animals are observed daily for signs of ill health, urine is collected, and urine protein is measured. Liver and kidney weights were recorded at the time of dissection. The liver and kidney were dissected from each animal, fixed, sectioned and stained with Sirius Red and H&E. A piece of each liver is used to measure hydroxyproline.

Hydroxyproline measurement: Liver and spleen hydroxyproline content was determined using a hydroxyproline colorimetric assay kit (BioVision, Milpitas, CA, USA).

Histology: After treatment, mice were anesthetized and perfused with PBS by intracardiac puncture. The liver and spleen are measured, and organ biopsies are fixed in 4% neutral buffered formalin, embedded in paraffin, and sectioned.

Example 5.17: Treatment and/or prevention of non-alcoholic

steatohepatitis (NASH)

The therapeutic efficacy of the compounds of the present invention was evaluated using the NASH mouse model as described by Barbara Ulmasov B. et al. (Hepatology Communications, 2018, 3(2) -https://doi.org/10.1002/hep4.1298).

Mice: 5-week-old C57BL/6J male mice were housed in standard facilities under controlled conditions of temperature, humidity, and a 12-h/12-h light/dark cycle with free access to water.

Choline deficiency, L-amino acid-defined, high-fat model of NASH: Choline-deficient, amino acid-defined, high-fat diet (CDAHFD)22 was obtained from Research Diets (A06071309; New Brunswick, NJ). This diet is formulated as a high-fat, choline-deficient diet that includes 0.1% methionine and 45% calories% fat (20% lard, 25% soybean oil). The control diet is standard rodent chow containing 13.6% calories from fat. Starting at 6 weeks of age, 30 mice are placed on CDAHFD and 30 mice on standard diet. At the end of 6 weeks, 10 mice from each diet group were starved for 5 hours and killed. Blood and liver samples are taken. Livers were divided into sections that were fixed in 10% phosphate-buffered formalin, frozen in liquid nitrogen, or placed in RNA stabilization solution for future evaluation. The remaining mice are kept on the diet for another 4 weeks, for a total of 10 weeks, and then starved, killed, and sampled.

Biochemical analysis: Liver triglyceride content was measured using a colorimetric triglyceride assay kit (Caiman Chemicals, Ann Arbor, MI) according to the manufacturer's instructions. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, insulin, triglyceride, and cholesterol levels were measured commercially by Advanced Veterinary Laboratories (Saint Louis, MO).

RT-PCR: Real-time reverse transcription polymerase chain reaction is performed to calculate changes in mRNA abundance.

Histopathology: Formalin-fixed liver sections were embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin (H&E) using a standard protocol for microscopic evaluation. To assess liver collagen content, paraffin-embedded liver sections were stained with Sirius Red/Fast Green. Sirius red binds to all types of collagen, while fast green stains non-collagenous proteins.

Liver sections were preprocessed to remove paraffin, and cores were counterstained using Weigert's iron hematoxylin solution. After washing, tissues were stained with 0.1% Sirius Red (Direct Red 80; Sigma, Saint Louis, MO), 0.1% Fast Green FCF Certified (Sigma) in saturated picric acid for 2 hours. Slides are then washed in water, dehydrated with ethanol and xylene, and finally mounted in Permaslip (Alban Scientific, Inc., Saint Louis, MO). The degree of collagen accumulation is assessed by morphometric analysis.

Determination of hydroxyproline content in the liver: Hydroxyproline content is determined as a measure of the amount of total collagen present in the liver. The liver tissue is homogenized in distilled water, precipitated with trichloroacetic acid and hydrolyzed for 48 hours in 12 N HCl at 105 °C. The samples are evaporated, and the dry pellets are reconstituted in distilled water. The reconstituted samples are centrifuged for 10 minutes at 13,000 g and the supernatants are diluted with 12 N HCl to achieve a concentration of 4 N HCl in the final samples. The content of hydroxyproline in the liver is determined using the sensitive tissue hydroxyproline test.

Immunohistochemistry: Formalin-fixed liver tissues were preprocessed to remove paraffin by standard methods. Endogenous peroxidase activity was blocked by incubation in 3.3% hydrogen peroxide in methanol for 1 hour at room temperature.

Detection of apoptotic cells in liver tissues: Apoptotic cells in liver tissues are identified by labeling and detection of DNA strand breaks by deoxyuridine triphosphate-mediated end-labeling (TUNEL) method using the ApopTag peroxidase detection kit (Millipore, Temecula, CA). Stained apoptotic cells with hepatocyte morphology were counted. To detect apoptotic cells with HSC morphology, TUNEL-stained liver tissues were subsequently stained with an antibody to desmin (an HSC marker), except that the secondary peroxidase activity of the antibody was detected using the ImmPACT VIP Peroxidase Substrate Kit (Vector Laboratories).

Western Blot: Western blot was performed as described using the following primary antibodies: anti-phosphorylated maternal anti-decapentaplegic homolog 3 (p-SMAD3), ab52903, 1:1000 (Abeam); anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH), sc-25778 (Santa Cruz Biotechnology, Inc., Dallas, TX).

Example 5.18: Treatment and/or Prevention of Diabetic Nephropathy or Diabetic Kidney Disease (DN or DKD)

The therapeutic efficacy of the compounds of the present invention was investigated in vivo using a mouse model of type I diabetes (STZ-induced diabetes).

Animal model: 10-week-old male 129/SV mice (Charles River, Germany) are housed in individually ventilated cages, on a standard diet with free access to tap water. Weight-matched 129/SV mice received either 125 mg/kg body weight STZ (Sigma-Aldrich) in 50 mM sodium citrate (pH 4.5) or sodium citrate buffer (nondiabetic control group) intraperitoneally on days 1 and 4 to induce hyperglycemia (hyperglycemia >

15 mmol/1). Mice do not receive insulin during the study.

Diabetic mice are given a placebo (saline solution) for 12 weeks or the test compounds. Body weight and glucose levels are measured weekly. HbA1c (Olympus AU400) and kidney function (serum creatinine) are measured at 6 and 12 weeks. Albuminuria is assessed using a mouse albumin ELISA kit. Sensory nerve conduction velocity (NCV) studies were performed in mice anesthetized with 2% isoflurane at weeks 6 and 12. Tail sensory NCV was determined by stimulation proximally along the tail at a recorded distance of 3 cm. Toennies Inc. neuro-screen is used for measurement. After 12 weeks of follow-up, mice were euthanized, bilateral thoracotomy and laparotomy were performed, and the kidneys were perfused with ice-cold saline via the left ventricle.

Immunohistochemistry: For immunofluorescence, paraformaldehyde-fixed and paraffin-embedded tissue sections (2 µm) are processed. After blocking with 10% rabbit serum, paraffin sections were stained with antibodies against pP38 and F4/80 and with a Cy3-conjugated secondary antibody.

Example 5.19: Treatment and/or Prevention of Diabetic Nephropathy, Diabetic Kidney Disease, and Diabetic Retinopathy in an STZ-Induced Diabetic Model.

A total of 70 female CD-1 mice aged 5-7 weeks, weighing approximately 25-30 g, were used for the study. They were purchased from Charles River, UK and had an acclimation period of 7 days. Animals were housed in IVC cages (up to 5 per cage) with individual mice identified by tail. All animals were allowed free access to a standard certified commercial diet and disinfection during the study. The holding room was maintained under standard conditions: 18-24 °C, 55-70% humidity and a 12-hour light/dark cycle.

All protocols used in this study were approved by the Animal Welfare and Ethical Review Committee and all procedures were carried out in accordance with the guidelines of the Animals (Scientific Procedures) Act 1986.

Mice (except the negative control group) were injected with STZ (55 mg/kg; IP) daily for 5 consecutive days. Mice were fasted for 6 hours before injection. One week after the end of induction glucose levels were monitored and mice with levels less than 15 mml/l (280 mg/gl) were removed from the study because they did not develop the ideal severity of diabetes. Glucose levels were monitored weekly, and 5 weeks after STZ induction, biochemical assessment of kidney damage was performed by measuring urinary albumin and creatinine levels and confirming that they were within the desired range. After the successful completion of this phase, the animals were assigned to treatment groups.

Table 53:

Treatment was continued for 4 weeks (28 days) with weekly blood glucose and urine albumin/creatinine measurements. The study schedule is detailed below, looking at weekly (weeks 5 to 9) blood glucose levels, urinary creatinine albumin ratio, and terminal (week 9) blood chemistry and cytokines, renal histology, and eye-retinal leak.

Table 54:

Results

Clinical signs: An initial decrease in body weight was observed for many treatment groups, which is to be expected and in most cases body weight gradually recovered by the end of the dosing period. Body weight of some animals fell below 90% of initial body weight, however no animal had to be rested for dosing.

Blood Glucose: Induction with STZ resulted in a significant increase in blood glucose compared to uninduced vehicle controls (Table 55). IVc-059a IV treatment resulted in a significant reduction** in blood glucose levels compared to vehicle-induced controls on day 28. Treatment with IIIc-061a IV also resulted in a reduction in blood glucose levels, but this did not reach statistical significance.

Table 55: Mean blood glucose concentration on day 0 and day 28 of female ICR-CD1 mice in the STZ-induced diabetes model. Values shown are mean ± SD; n=3 for uninduced and induced vehicle groups; n=8 for all other treatment groups.

Kidney Weight: There was no statistical difference in either wet kidney weight or kidney weight as a percentage of animal body weight.

Urine Albumin: Creatinine (ALB: CRE): Urine was collected weekly and pooled for each treatment group. Treatment with Ib-010a, IIIc-061a and IVc-059a resulted in a decrease in ALB:CREA (Table 56) on the last day of treatment on the 28th before sacrifice (week 9 of the schedule).

Blood urea nitrogen (BUN): Treatment with Ic-007, Ib-010a, IIIc-061a, and IVc-059a resulted in blood urea nitrogen levels that were significantly lower than induced vehicle controls.

ELISA: At the end of the treatment period, blood was collected and processed into serum. Serum levels of CRP, TGF-β, IL-6 and TNF-α were quantified using commercially available ELISA kits, the results are shown in Table 56.

Serum CRP: Serum CRP levels in female ICR-CD1 mice in the STZ-induced diabetes model after 28 days of treatment. Serum CRP levels were significantly increased in STZ-challenged animals. Treatment with Ia-001a, Ia-001aTz and control captopril resulted in significantly reduced serum CRP levels compared to vehicle-induced controls (**p=0.0074, *p=0.0304 and **p=0.0026 respectively, One-way ANOVA, Table 56).

Serum TGF-β: Serum TGF-β levels were significantly elevated in STZ-challenged controls. Treatment with all compounds except Ic-007a and IIIc-061a resulted in TGF-β levels that were significantly lower than vehicle-induced controls (p≤0.05; One-way ANOVA, Table 56).

Serum IL-6 levels were significantly elevated in STZ-challenged controls. Treatment with all compounds except IVc-059a and the control compound captopril resulted in IL-6 levels that were significantly lower than vehicle-induced controls (p≤0.05; One-way ANOVA, Table 56).

Serum TNF-α levels were significantly elevated in STZ-challenged controls. Treatment with Ia-001a, Ia-001aTz, Ic-001aTz/1-004a, and IIIc-061a resulted in TNF-α levels that were significantly lower than vehicle-induced controls (p≤0.05; One-way ANOVA, Table 56).

Kidney histology: Collagen quantification was performed at the end of the dosing period, animals were killed and the kidney was resected. The tissue was fixed in formalin and embedded in paraffin wax, then stained with Masson's trichrome according to the manufacturer's instructions. Collagen was quantified using the published method by Chen et al., 2017. Collagen coverage was significantly increased in STZ-induced vehicle controls compared to uninduced vehicle controls. Treatment with IIIc-061a and IVc-059a resulted in significantly reduced collagen levels compared to induced vehicle controls (p=0.0210 and p=0.0134 respectively; One-way ANOVA, Table 56).

Disease Score: Renal disease score was quantified using H&E staining and assessment of mesangial and glomerulosclerosis, arteriolar hyalinosis, and GBM thickening. Apparently, the disease score was significantly increased in STZ-induced vehicle controls compared to non-induced vehicle controls. Treatment with all compounds except Ic-001aTz/1-004a resulted in a significantly reduced disease score compared to vehicle-induced controls (p≤0.005; One-way ANOVA, Table 56).

Retinal Histology: At the end of the dosing period, immediately before terminal sampling, 4 animals per group were injected with FITC-dextran to evaluate retinal flat surfaces and the animals were killed and the eyes resected.

Retinal flat sections were prepared and imaged using a fluorescence microscope (EVOS, Thermo Fisher). The area covered by the vasculature was significantly increased in STZ-induced vehicle controls compared with non-induced vehicle controls. Treatment with Ic-007a, IIIc-061a and IVc-059a resulted in significantly reduced vasculature coverage compared to induced vehicle controls (p=0.0432, p=0.0337 and p=0.0131 respectively; One-way ANOVA, Table 56).

Table 56: Urine Creatinine Albumin Ratio (ALB:CRE), Blood-BUN, Blood-CRP, Blood-TGF-beta, Blood-IL-6, Blood-TNF-alpha, Trichrome Stain (Renal Fibrosis), Disease Score, and Eye Area by Day 28 of Treatment (Week 9 Schedule)

Example 5.20: Treatment and/or prevention of polycystic kidney disease (PKD or PCKD)

The therapeutic efficacy of the compounds of the present invention was investigated in vivo using the Pci mouse model as described by Lee EC et al. (Nat Commun 10, 4148 (2019).

Preclinical model: the pci mouse, a genetically relevant PKD rodent model closely correlated with human disease for use in evaluating new agent efficacy. This model develops PKD linked to the gene that causes the human disease, providing a transmissible rodent model that closely mirrors the development of PKD in humans.

It has been extensively characterized for disease progression and response to treatment, providing a high level of confidence for drug discovery studies.

Animal model: pci mouse, CD-1-pci<Iusm>strain (CrownBio) carrying a gene mutation associated with the same gene that causes human nephronophthisis type 3. Symptoms and disease progression is a slowly progressive renal cystic disease with cysts that develop in the collecting tubules and other segments of the nephron becoming cystic as the disease progresses. Male and female mice are similarly affected by the disease. Mice are 5 weeks old and receive either normal diet, normal diet with vehicle (negative control), or positive control and test compound, all mixed with normal diet. Controls: the vehicle used for the compounds was used as a negative control in one group of animals, and the vasopressin-2 (V2) receptor antagonist tolvaptan was used as a positive control in one group of animals.

Output and endpoints: Cyst and fibrosis extent in the kidney, including histological verification, kidney weight, serum BUN, blood test article concentration, body weight, and food intake were recorded weekly.

Example 5.21: Treatment and/or Prevention of Radiation-Induced Fibrosis (RIF)

The therapeutic efficacy of the compounds of the present invention was investigated in vivo using the RIF mouse model as described by Ryu SH et al. (Oncotarget.

2016, 7(13), 15554-15565, doi:10.18632/oncotarget.6952).

Radiation-induced fibrosis (RIF) is one of the most common late complications of radiation therapy. In the RIF mouse model, the leg contracture test is used to test the in vivo efficacy of the compound.

Radiation-induced fibrosis (RIF) is characterized by excessive accumulation of extracellular matrix in the skin and soft tissues, and fibroblast proliferation is one of the most common late complications of radiation therapy. Also, RIF is an irreversible process of dead fibrous tissue and a dynamic process related to scar tissue remodeling by reactivated myofibroblasts.

RIF mouse model: Male BALB/c mice are used for the RIF mouse model. Under anesthesia, the right hind limb of each mouse received radiation doses of 44 Gy (22 Gy × 2 times over 2 weeks) using a linear accelerator. A specially designed shield is used to protect the rest of the body, and a 1 cm thick bolus is applied over the skin to provide an adequate dose of radiation to the surface of the hind leg. After irradiation, the mice were randomly divided into two groups. Each group will be treated once a day with saline (control group) or compounds (treated group). Mice that did not receive radiation or drug were used as a negative control group.

To demonstrate the antifibrotic effect of the compounds in the skin and soft tissue of irradiated legs, the thickness of the epithelium from the surface of the epidermis to the base of the dermis is measured by H&E staining.

Example 5.22: Treatment and/or prevention of Stargardt disease (SD)

The therapeutic efficacy of the compounds of the present invention was investigated in vivo using the SD mouse model as described by Fang I. et al. (Facebook Journal, 2020, DOI: 10.1096/fj.201901784RR).

Animals: Mice Pigmented Abca4-/- mice (129S4/SvJae-Abca4tm1Ght) will be purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Pigmented wild-type (VT) mice (129S2/SvPas) were obtained from Janvier Labs (Le Genest-Saint-Isle, France). In each group, the ratio of males to females is 1:1. Mice were bred and housed under a 12:12h light cycle (approximately 50 lux in cages) with food and water ad libitum.

Blue light illumination (BLI): Age-matched (9 months) pigmented Abca4-/- and VT mice were anesthetized intraperitoneally with a three-component anesthetic consisting of 0.05 mg/kg fentanyl, 5 mg/kg midazolam, and 0.5 mg/kg medetomidine. The pupils were dilated with a mixture of 0.5% tropicamide and 2.5% phenylephrine hydrochloride. METHOCEL (Omni Vision, Puchheim, Germany) is applied to moisten the cornea. During light exposure, a glass is placed on the cornea of the exposed eye. The illuminated eye of each mouse was exposed to blue light (wavelength: 430-nm) at an intensity of 50 mV/cm2 for 15 minutes. The unilluminated control eye was carefully covered to protect it from stray light.

A series of tests are performed including optical coherence tomography (OCT), light microscopy and transmission electron microscopy (TEM), bisretinoid quantification by HPLC, and full-field electroretinography ERG before and seven days after BLI.

Example 5.23: Treatment and/or prevention of proliferative

vitreoretinopathy (PVR)

The therapeutic efficacy of the compounds of the present invention was investigated in vivo using the PVR mouse model as described by Hou H et al. (Ophthalmic Res 2018; 60: 195-204. doi: 10.1159/000488492) or by Márkus B. et al. (FEBS Open Bio.2017 (7) / 8 (1166-1177) Published June 29, 2017 doi: 10,1002/2211-5463,12252).

Animal models: A murine model of PVR is used by intravitreal injection of the proteolytic enzyme, dispase. This model is known to induce glial activation as well as epi- and subretinal membrane formation.

4- to 6-month-old wild-type females are anesthetized with pentobarbital (90 mg·kg-1, ip) and also receive one drop of 1% procaine hydrochloride (Novocaine, EGIS, Budapest, Hungary) for topical anesthesia and one drop of tropicamide (Mydrum, Chauvin Aubeans, Montpellier, France) for iris dilation. Four microliters of dispase (Sigma-Aldrich; 0.4 U·µL-1, dissolved in sterile saline) was injected intravitreally into the right eyes under stereomicroscopic control (Ctrl) using an automatic pipette. Control animals received 4 µl of sterile saline. Stratus optical coherence tomography (OCT; Carl Zeiss Meditec, Dublin, CA, USA) scans are taken after injections to confirm PVR induction and monitor disease progression. Mice treated with Ctrl and dispase were sacrificed on day 14 after injections when signs of PVR formation were evident: presence of epiretinal membrane and/or retinal detachment on OCT examination.

Specimen preparation and identification: The vitreous bodies of mice are isolated after guillotine removal of the cornea along with the scleral gall using a scalpel blade, followed by removal of the lens. After vitreous solubilization with lysis buffer (pH 8.5) containing 7 m urea, 30 m m Tris, 2 m thiourea, and 4% CHAPS, lysates were sonicated in an ice-cold water bath for 5 min and centrifuged at 16,900 g for 10 min at 4 °C. Supernatants are transferred to LoBind Eppendorf tubes and purified using the Readi-Prep 2-D CleanUp kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's protocol.

Briefly, after precipitation and centrifugation, the pellets are dried and resuspended in 240 µl rehydration buffer containing 7 m urea, 2 m thiourea, 4% w/v.

CHAPS, 1% dithiothreitol (DTT), 2% vol./vol. Bio-Lite Bio-Rad) and 0.001% bromophenol blue, and immediately used for isoelectric focusing.

2D gel electrophoresis: Three vitreous samples from each group were subjected to 2-DE. First immobilized pH gradient (IPG) strips with IPG (24 cm, pH 4-7; Bio-Rad) were rehydrated with extracted vitreous proteins using passive rehydration at 20 °C overnight. This is followed by isoelectric focusing performed by applying 300 V for 3 h, which is gradually increased to 3500 V for 5 h and then held at 3500 V for 18 h. After isoelectric focusing, the IPG strips were immediately placed at -70 °C until equilibration. IPG strips were equilibrated for 15 min in equilibration buffer (500 m m Tris/HCl, pH 8.5, 6 m urea, 2% SDS, 20% glycerol) containing 0.6% DTT and for 15 min in equilibration buffer containing 1.2% id. In the second dimension, strips are placed on top of 12% polyacrylamide gels and covered with agarose. Using a Protean Plus Dodeca Cell (Bio-Rad) electrophoresis was performed at 100 mA per gel for 24 h until the bromophenol blue dye reached the bottom of the gel. All 12 gels work together under the same conditions. Proteins were stained using ruthenium II tris(bathophenanthroline disulfonate) fluorescent dye 25 prepared in house, and gel images were captured using a Pharos FKS Plus molecular Imager (Bio-Rad). In all cases, three biological replicates are analyzed, samples from the Ctrl and dispase-treated groups are processed together on the same day.

Example 5.24: Treatment and/or Prevention of Wet and Dry Age-Related Macular Degeneration (ARMD or AMD)

The therapeutic efficacy of the compounds of the present invention was investigated in vivo using an AMD mouse model as described by Matsuda Y et al. (Mol Ther Nucleic Acids.2019;17:819-828. doi:10.1016/j.omtn.2019.07.018).

Animals: Female C57BL/6J mice were obtained from Charles River Laboratories Japan. Male BN rats are obtained. Male New Zealand White (NZW) rabbits were obtained from Kitayama Labes, Japan. Mice, rats and rabbits are maintained in special pathogen-free conditions.

EMT analysis of RPE cells: RPE cells cultured at 37 °C were seeded at 1 × 105 cells/well in 96-well microtiter culture plates. After incubation for 24 h, cells were treated with FGF2 (2 ng/ml) and TGF-β2 (3 ng/ml) with and without compounds of the present invention for 3 days. The mRNA expression levels of the EMT biomarkers α-SMA and collagen type I were assessed by quantitative real-time PCR.

Matrigel Plug Assay: 0.5 ml of Matrigel Matrix GFR (reduced growth factor)-PRF (phenol red free) solution (BD Biosciences) is mixed with 1 µg FGF2 and implanted subcutaneously into the right flank of female C57BL/6J mice (8 weeks, n = 3. Compounds of the present invention are administered intraperitoneally, and Matrigel plugs are removed and photographed on day 7 post-implantation. angiogenesis was determined by the concentration of hemoglobin in the Matrigel plug, using the cyanmethemoglobin method according to the manufacturer's instructions.The optical density values of the samples at 540 nm (OD540) were measured in a microplate reader, and the hemoglobin concentrations were calculated according to hemoglobin standards (Sigma-Aldrich).

Laser-induced CNV mouse models: Midrin-P ophthalmic solution is instilled into the eyes of male C57BL/6J mice to dilate the pupils. Then, laser irradiation (wavelength, 532 nm; spot size, 50 µm; irradiation time, 0.1 s; laser output, 120 mV) was applied to 6 sites of the eye using a slit lamp (SL-130) and a multicolor laser photocoagulator (MC-300), avoiding large retinal capillaries. Immediately after the induction of CNV by laser irradiation, the test compounds according to the invention and the control are administered by intravitreal injection. Seven days after laser irradiation, a 4% solution of FITC-dextran is administered into the tail vein in a volume of 0.5 ml/animal. One to five minutes after FITC-dextran administration, animals were euthanized by cervical dislocation. Eyeballs are removed and fixed in 4% paraformaldehyde-phosphate buffer for 12-24 h. Choroid flat mounts are prepared under a stereoscopic microscope. Photographs of CNV sites are taken using a confocal microscope. During laser-induced CNV experiments in animals, successful irradiation was confirmed for each irradiation as the formation of air bubbles in the eye of animal models.

Rat models of laser-induced CNV and subretinal fibrosis: The same technique will be used in a rat model using male BN rats. For the CNV model, the laser settings are a spot size of 80 µm and an irradiation time of 0.05 s at 120 mV at 8 sites in the retina-choroid. For the CNV model of subretinal fibrosis, the laser power used is 240 mV. Immediately after laser irradiation, an intravitreal injection with control and test compounds according to the present invention is performed. For subretinal fibrosis CNV studies, (1) saline vehicle and (2) compounds of the present invention (15 µg/eye for a single injection or 2 or 3 injections at 2-week intervals) are used. After laser irradiation, for CNV studies, the FITC-dextran protocol as above is used 14 days after laser irradiation, with preparation of enucleated eyes for flat choroid confocal microscopy. For subretinal fibrosis studies, after 6 weeks, animals were sacrificed and enucleated eyes were prepared for histopathological studies to quantify subretinal fibrosis using light microscopy. Eyes were embedded in paraffin blocks and sectioned and stained with Masson trichrome according to routine methods. Fibrosis is graded blindly by two or three independent investigators according to the following scale: grade 0, none; grade 1, minimal; grade 2, mild; grade 3, moderate; grade 4, difficult.

Example 5.25: Treatment and/or prevention of pterygium (PTE)

The therapeutic efficacy of the compounds of the present invention was evaluated on choroidal angiogenesis and pterygium growth using a choroidal neovascular mouse model (CNV), a direct in vivo angiogenesis assay (DIVAA) and a pterygium mouse model.

Pterygium is a benign fibrovascular growth of the ocular surface that is usually associated with discomfort and red eyes, and as the disease progresses it is often associated with reduced vision (topographic astigmatism) and restriction of ocular motility in severe cases. A wide range of pro-inflammatory cytokines induced by fibrogenic growth factors, oxidative stress and DNA methylation have been implicated in the pathogenesis of pterygium. Because some of these factors are affected by exposure to ultraviolet (UV) light, current evidence from multiple sources suggests that individuals with high exposure to sunlight are at increased risk for pterygium. Despite extensive research, there is no clear understanding of the pathogenesis of pterygium, but one of the most important factors contributing to the pathogenesis is the neoplastic changes of limbal stem cells associated with exposure to UV light and the possible role of an oncogenic virus (human papillomavirus).

To date, three animal models of pterygium have been described, using injection of human pterygium epithelial cells, exogenous extracellular matrix, or UV scattered radiation in rabbits and mice. The results of the rabbit model using UV scattered light focused on computational prediction of the size and shape of tissue growth, without histological analysis. The mouse model showed the histological characteristics of human pterygium, however, manipulation of rabbits for ophthalmic procedures is easier since the structure and size of the eye more closely resembles that of humans.

Procedures: Mice received water with or without test compounds. For CNV, neovascular lesion volume was determined in choroidal retinal pigment epithelium (RPE) flat mounts using confocal microscopy seven days after laser induction. For DIVAA, silicone capsules containing 10,000 human pterygium epithelial cells are implanted in the flanks of mice subcutaneously. After eleven days, neovascularization (NV) was quantified by spectrofluorimetry after injection of fluorescein isothiocyanate (FITC)-labeled dextran into the mouse tail vein. A pterygium epithelial cell model was developed by injecting 10,000 human pterygium epithelial cells into the nasal subconjunctival space of athymic nude mice.

Main Outcome Measures: Student's t-test is used to evaluate data for CNV and DIVAA models, and histological preparations are used to evaluate pterygial lesions.

Example 5.26: Treatment and/or prevention of central serous chorioretinopathy (CSC)

The therapeutic efficacy of the compounds of the present invention was investigated in vivo using a CSC animal model as described by Wei-Da Chio, et al., Life Sci J 2019;16(12): 115-126 ISSN: 1097-8135

The pathogenesis of central serous chorioretinopathy (CSC) is still not fully understood. The involvement of corticosteroids is indisputable, although theirs

exact role not clarified; other parts of the underlying mechanism of CSC are mainly elucidated by imaging techniques such as fluorescein and indocyanine green angiography. Although most cases of CSC are self-limiting, there are severe and recurrent courses, and only a limited number of treatment options are available for these patients: laser photocoagulation, with the risk of scotoma and choroidal neovascularization, and photodynamic therapy.

Procedure: Treatment was performed with male SD rats with CSCR. The right eyes of the cases were randomly divided into 2 groups (control and test compound) and underwent a series of examinations including indirect ophthalmoscopy and OCT scanning every week. All SD rats are first introduced to the CSCR model. If CSCR disappears under OCT imaging, the compound is considered effective.

Example 5.27: Treatment and/or prevention of cystic fibrosis (CF) affecting the lungs, but also the pancreas, liver, kidneys and intestines

The therapeutic efficacy of the compounds of the present invention was investigated in vivo using a CF mouse model as described by McCarron A et al. (Respir Res.2018 (19) / 1 (54) Published April 2, 2018 doi: 10.1186/s 12931-0 18-0750-i)

In humans, cystic fibrosis (CF) lung disease is characterized by chronic infection, inflammation, airway remodeling, and mucus obstruction. The lack of pulmonary manifestations in CF mouse models hinders research into the pathogenesis of airway diseases, as well as the development and testing of potential therapeutics. However, recently generated CF animal models, including rat, ferret, and pig models, exhibit a variety of well-characterized lung disease phenotypes with varying degrees of severity. Various airway phenotypes of currently available CF animal models are available and represent the potential application of each model in airway-related CF research. Specifically, mutant alleles with common human CF-causing mutations (eg, Phe508del or Gli551Asp) introduced into the mouse CFTR sequence have been developed.

Example 5.28: Treatment and/or Prevention of Idiopathic Pulmonary Fibrosis (IPF)

The therapeutic efficacy of the compounds of the present invention was investigated in vivo using an IPF animal model as described by Chen SS et al. (J Thorac Dis.2017;9(1):96-105. doi: 10.21037/jtd.22.01.2017.).

IPF is a chronic progressive interstitial lung disease with severe pulmonary fibrosis. The etiology of IPF remains unclear. Patients usually only survive 2-3 years after being diagnosed with IPF. The incidence and development of IPF are associated with epithelial cell injury, fibrocyte proliferation, inflammatory reactions, and extracellular matrix deposition. The pathological manifestation of IPF is common interstitial pneumonia (IIP). Currently, the mortality of patients with IPF is quite high. However, effective treatments for IPF are lacking. The leading cause of IPF-related death is acute exacerbation of IPF (AE-IPF), accounting for more than 50% of IPF-related deaths. Most patients with AE-IPF cannot tolerate bronchoscopy or lung biopsy because of their critical condition.

The lack of AE-IPF biopsy significantly limits in-depth research into AE-IPF. Experimental animal models that could mimic AE-IPF would be useful tools to study AE-IPF.

Animal models of bleomycin-induced IPF (BLM) are used.

A rat model of IPF was developed by intratracheal perfusion with BLM. Theoretically, a second intratracheal perfusion with BLM should cause additional lung injury in rats that already develop pulmonary fibrosis from the first perfusion. Additional lung injury may resemble the pathologic features of AE-IPF.

Sprague Dawley (SD) rats were randomized into different groups: control group treated with compounds alone, AE-IPF model group with compounds BLM BLM group, AE-IPF model group (cpd BLM BLM group), IPF model group treated with compounds (Cpd BLM group), IPF model group (BLM group) and normal control group without BLM.

Rats in the BLM BLM groups undergo a second perfusion with BLM on day 28 after the first perfusion with BLM. Rats in the other two groups received saline as the second perfusion. Six rats in each group were sacrificed on day 31, day 35, and day 42 after the first perfusion, respectively.

The pathophysiological characteristics of rats in the BLM BLM group resemble those of AE-IPF patients and as described (Chen SS, J Thorac Dis 2017; 9(1):96-105), second perfusion with BLM appears to cause acute worsening of pulmonary fibrosis and can be used to model AE-IPF in rats. The effects of compound formulations in this rat model using AE-IPF induced using one or two intratracheal bleomycin perfusions, respectively, should improve histopathological findings (H&E- and Masson-stained sections analyzed and scored for acute lung injury) as well as survival.

Example 5.29: Bleomycin-induced pulmonary fibrosis in 21-day-old C57BL/6 mice

A total of 40 male Balb/c mice aged 8-10 weeks were used for the study. They were purchased from Charles River, UK and had an acclimation period of 7 days. Animals were housed in IVC cages (up to 5 per cage) with individual mice identified by tail. All animals were allowed free access to a standard certified commercial diet and disinfection during the study. The holding room was maintained under standard conditions: 18-24 °C, 55-70% humidity and a 12-hour light/dark cycle.

All protocols used in this study were approved by the Animal Welfare and Ethical Review Committee and all procedures were carried out in accordance with the guidelines of the Animals (Scientific Procedures) Act 1986.

Mice were anesthetized using an IP injection of xylazine and ketamine.

Once the appropriate level of anesthesia was achieved, animals were infused with 1.0 U/kg bleomycin sulfate by intratracheal route. The total volume of liquid was 50 µl. Mice were randomly assigned to treatment groups as shown in Table 57 below:

Table 57:

IV dosing and positive control (pirfenidone) started 24 hours after bleomycin instillation.

The drug solution was formulated immediately before dosing. For a dose of 15 mg/kg (enough for 12.30 g mice), 4.5 mg of compound was weighed. 75 µl (5%) DMSO was added and mixed until the drug was dissolved. 150 µl (10%) solution was added and gently mixed, 150 µl (10%) PEG400 was added and gently mixed. 1000 µl PBS (pH7) was added and the matrix was weighed to ensure maintenance of pH 7.0. The remaining PBS was added (up to a total of 1500 ul). The compound remained in solution.

Post-completion sampling (final day of dosing): On the day of sampling, 2 hours after the final dose, mice were sacrificed with an overdose of anesthesia. Animals were sampled for the following:

Serum: Immediately after sacrifice, blood was collected by cardiac puncture and placed in Eppendorf tubes. The tubes were stored at room temperature for at least 20 minutes before centrifugation at 6,000 rpm for 5 minutes. The samples were stored at -80 °C.

Gross necropsy: After cutting, the internal organs are exposed and observed. Any abnormalities were noted.

Lungs: Lungs were inflated with formalin before resection. Briefly, the trachea of the mouse was exposed, and a small incision was made using a blade. Suture material is gently placed around the back of the trachea below the incision and loosely tied. Formalin was gently instilled into the lung and the suture was tightly closed to ensure inflation. Lungs were fixed in formalin and stained with trichrome and H&E stain for analysis of collagen deposition and disease progression (Ashcroft score). Part of the lung tissue was suddenly frozen.

Ashcroft scoring:

0 Normal lungs

1 Minimal fibrous thickening of alveolar or bronchiolar vessels

2 Between minimal and moderate

3 Moderate thickening of the walls without obvious damage to the lung architecture

4 Between moderate and increased fibrosis with damage to the lung structure

5 Increased fibrosis with definite damage to the lung structure and the formation of fibrous bands or small fibrous masses

6 Between increased fibrosis and severe distortion of lung structure

7 Severe structural distortion and large fibrous areas;

"lungs in a honeycomb"

8 Total fibrous obliteration of the field

Results

Clinical signs: No changes in body weight were noted and no animals required a dosing break during the study.

Ashcroft score: Lungs from each animal were FFPE, sectioned and stained with H&E according to the in-house protocol. Slides were captured on a Glissando slide scanner to obtain whole slide images. Each lung was scored using the Ashcroft scoring system for the degree of inflammation/fibrosis according to Hübner et al., 2008. Scoring was performed in a blinded fashion. And pirfenidone in a dose of

100 mg/kg and IVc-059a at 15 mg/kg reduced the onset of fibrosis in mouse lungs (p=0.0018 and 0.0130, respectively, ANOVA). Pirfenidone treatment had the same effect on the onset of fibrosis as IVc-059a treatment (p=0.4441, 2-tailed T-test).

Representative images are shown in Figure 11A.

Figure 11A shows representative images of lung fibrosis on day 21 of treatment: Examples of connective tissue trichrome staining images (x20); (A1) uninduced (healthy); A2, Bleomycin induced by vehicle treatment; A3, Bleomycin induced by 100 mg/kg pirfenidone treatment; A4, Bleomycin induced by IVc-059a treatment. Figure 11B shows the Ashcroft score of healthy, untreated bleomycin versus pirfenidone versus IVc-059a.

A whole lung from each animal was formalin-fixed and stored in 70% ethanol. These sections were dehydrated in increasing concentrations of ethanol before being embedded in paraffin wax. Sections were cut at 5 µm and baked on Superfrost slides for 2 hours. Sections were stained using a commercially available connective tissue kit (Abeam ab150686). The percentage of area covered by trichrome staining was quantified using Kupath and ImageJ software.

Example 5.30: In vitro results and ADME toxicology

A comprehensive ADME-toxicology evaluation was performed on compounds Ic-007a, IIc-007a, IIIc-061a and IVc-059a.

Cardiac Ion Channel Assay (CiPA) The Core QPatch Panel was evaluated for Ic-007a, IIc-007a, IIIc-061a and IVc-059a: The following three assays were used for CiPA: Nav1.5 QPatch CiPA assay on sodium ion channel cells, hERG QPatch CiPA assay on potassium ion channel cells, Cav1.2 (L type) QPatch CiPA assay on cells with human ion channels.

Hepatocyte microsomes and human hepatocyte metabolism were evaluated. A comprehensive set of binding, enzyme and uptake assays, as well as solution properties, in vitro absorption, metabolism and toxicity were evaluated. Gebnetic toxicity was assessed using the AMES* and micronucleus assays.

*Ames is a bacterial test for identifying compounds that can produce gene mutation and shows high predictive value in rodent carcinogenicity tests. In vitro

** In vitro micronucleus assays used to identify compounds that cause chromosome damage (clastogenic and aneugenic)

Results

Highly polar compounds show low metabolism in liver microsomes with a half-life longer than 60 min. No significant toxic effect on hepatocyte cells was observed in terms of viability. Most negatively charged compounds are highly bound to plasma proteins. No genotoxic effect was observed.

The compounds show no significant effect on Ames fluctuation tests* TA100 -S9, TA100 S9, TA1535 - S9, TA1535 S9, TA1537 - S9, TA1537 S9, TA98 -S9, TA98 S9 in concentration from 5 µm to 0.1 mm. No toxicity to bacteria was observed on TA100 -S9, TA1535 - S9, TA1537 - S9, TA98 - S9.

No effects were observed in the micronucleus assay for the range of concentrations evaluated from 8 µm to 1 mm. In the CiPA panel, three cell-based assays were used. Cav1.2 (L-type) Automated Cell-Clamp CiPA Assay Based on Cell Ion Channels, hERG Automated CiPA Assay on Cellular Potassium Channels, and Nav1.5 CiPA Assay with Automated Patch Clamp Cell-Based Human Ion Channels: All results were evaluated at 3 concentration levels and no significant inhibition was observed between 0.1 µm and 10 µm.

In the safety panel test, the compounds had no or very low inhibitory effects on the following enzyme systems showing a good safety profile on the human 5-HT transporter (h) (antagonistic radioligand); 5-HT1A (h) (agonist radioligand); 5-HT1B (h) (antagonist radioligand); 5-HT2A (h) (agonist radioligand); 5-HT2B (h) (agonist radioligand); 5-HT3 (h) (antagonist radioligand); A2A (h) (agonist radioligand); acetylcholinesterase (h); alpha 1A (h) (antagonist radioligand); alpha 2A (h) (antagonist radioligand); AR(h) (agonist radioligand); beta 1 (h) (agonist radioligand); beta 2 (h) (antagonist radioligand); BZD (central) (agonist radioligand); Ca2+ channel (L, dihydropyridine site) (antagonist radioligand); CB1 (h) (agonist radioligand); CB2 (h) (agonist radioligand); CCK1 (CCKA) (h) (agonist radioligand); D1 (h) (antagonist radioligand); D2S (h) (agonist radioligand); delta (DOP) (h) (agonist radioligand); dopamine transporter (h) (antagonist radioligand); ETA (h) (agonist radioligand); GR (h) (agonist radioligand); H1 (h) (antagonist radioligand); H2 (h) (antagonist radioligand); kappa (h) (KOP) (agonist radioligand); KV channel (antagonist radioligand); Lck kinase (h); M1 (h) (antagonist radioligand); M2 (h) (antagonist radioligand); M3 (h) (antagonist radioligand); MAO-A (radioligand antagonist); mu (MOP) (h) (agonist radioligand); N neuronal alpha 4beta 2 (h) (agonist radioligand); Na+ channel (site 2) (antagonist radioligand); nmDA (radioligand antagonist); norepinephrine transporter (h) (antagonist radioligand); PDE3A (h); PDE4D2 (h); Potassium channel hERG (human)- [3H] dofetilide; V1a (h) (agonist radioligand).

The anti-inflammatory effect of the compounds was consistent with partial inhibition of cyclooxygenase enzyme, for example Ic-007a, IIc-007a, IIIc-061a and IVc-059a showed partial inhibition of COX2 (cyclooxygenase-2).

Example 5.31: In vitro profiling with BioMap Plus

A series of in vitro tests were performed using primary human cell-based assays that model complex tissue and organ disease biology (vasculature, immune system, skin, lung) and general tissue biology for phenotypic profiling of compounds under conditions that preserve crosstalk and feedback mechanisms. relevant to in vivo outcomes.

The potency, selectivity, safety, mechanism of action and disease indication of compounds Ic-007a, IIc-007a, IIIc-061a, IVc-059a were evaluated at concentrations of 30 µm, 10 µm, 3.3 µm, 1.1 µm on the following cell systems using the BioMaps Plus Phenotypic Cell Assay (Eurofins-DiscoverX, San Francisco, CA, USA):

- Venular endothelial cells for evaluation of cardiovascular diseases, chronic inflammation, asthma, allergies, autoimmune diseases, atopic diseases

− PBMC with venular endothelial cells for evaluation of cardiovascular diseases, chronic inflammation, autoimmune diseases, chronic inflammation;

- B cells with PBMC for evaluation of autoimmune disease, inflammation;

- Bronchial epithelial cells for evaluation of pneumonia, COPD

- Coronary artery smooth muscle cells for evaluation of cardiovascular inflammation, restenosis

- Dermal fibroblasts for evaluation of fibrosis, chronic inflammation, wound healing, tissue remodeling, matrix modulation

- Keratinocytes/dermal fibroblasts for assessment of psoriasis, dermatitis, skin biology

- Lung fibroblasts for evaluation of fibrosis, chronic inflammation, matrix remodeling

- Venular endothelial cells with macrophages for evaluation of cardiovascular inflammation, restenosis, chronic inflammation

BioMpas Plus references: Book: Phenotypic Drug Discovery, Chapter 2 - Development and Validation of Disease Assays for Phenotypic Screening. Ellen L. Berg, Sheryl P. Denker and Alison O'Mahony. https://doi.org/10.1039/9781839160721-00020 ISBN 978-1-83916-072-1

Assessing bioactivity-exposure profiles of fruit and vegetable extracts in the BioMAP profiling system. Wetmore BA, Clewell RA, Cholewa B, Parks B, Pendse SN, Black MB, Mansouri K, Haider S, Berg EL, Judson RS, Houck KA, Martin M, Clewell HJ 3rd, Andersen ME, Thomas RS, McMullen PD. Toxicology in Vitro. 2019,54,41-57.

Results

Ic-007a was not cytotoxic at the concentrations tested in this study. Ic-007a was antiproliferative for human primary T cells (30 µm) and fibroblasts (30 µm, 10 µm, 3.3 µm, 1.1 µm). Ic-007a was active with 20 labeled reads, and mediated changes in key biomarker activities are listed by biological and disease classifications. Mainly, Ic-007a affected activities related to inflammation (decreased VCAM-1, sTNFα, MIP-1α, I-TAC, MIG; increased IL-8; modulated IP-10), immunomodulatory activities (decreased CD40, M-CSF) and tissue remodeling activities (decreased collagen I, collagen IV, TIMP-1, tPA, collagen III, PAI-1, uPAR, bFGF).

Ic-007a was not cytotoxic at the concentrations tested in this study. IIc-007a was not cytotoxic at the concentrations tested in this study.

IIc-007a was active with 12 labeled reads, and mediated changes in key biomarker activities are listed by biological and disease classifications. Mainly, IIc-007a affected the activities related to inflammation (decreased sTNFα, MIP-1α; increased IL-8; modulated MCP-1), immunomodulatory activities (decreased sIL-10, sIL-2) and tissue remodeling activities (decreased collagen I, collagen IV, collagen III, PAI-1).

IIIc-061a was not cytotoxic at the concentrations tested in this study. IIIc-061a did not have any antiproliferative effect on these human primary cells at the concentrations tested. IIIc-061a was active with six labeled reads, and the mediated changes in key biomarker activities are listed by biological and disease classifications. IIIc-061a affected inflammation-related activities (reduction of I-TAC, MIG), immunomodulatory activities (reduction of sIL-17A), and tissue remodeling activities (reduction of collagen I, collagen III, PAI-1).

IVc-059a was not cytotoxic at the concentrations tested in this study. IVc-059a was antiproliferative for human primary endothelial cells (1.1 µm). IVc-059a was active with three labeled readouts, and mediated changes in key biomarker activities are listed by biological and disease classifications. IVc-059a affected inflammation-related activities (reduction of sTNFα) and immunomodulatory activities (reduction of sIL-10, sIL-2).

Example 5.32: In Vitro Results on Human Retinal Pigment Epithelial (RPE) ARPE-19 and Human Retinal Endothelial Cells (HRECs)

Compounds Ic-007a, IIc-007a, IVc-059a and bevacizumab (BEVA)) were evaluated for their effect on the disruption of the inner and outer blood retinal barriers caused by diabetic conditions. Two different concentrations of compounds were evaluated at 25 µm and 50 µm Ic-007a, IIc-007a, IVc-059a. Mechanisms of action involved in the anti-permeability effect of the tested treatments were investigated by assessing the permeability for the production of fluorescent dextran and pro-inflammatory cytokines using their mRNA expression levels by RT-PCR.

Outer blood retinal barrier conditions: human retinal pigment epithelial (RPE) cell cultures ARPE-19, a spontaneously immortalized human RPE cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). ARPE-19 cells were also grown under euglycemic conditions (D-glucose (D-Glc), 5.5 mmol/L) and hyperglycemic conditions (D-glucose, 25 mmol/l) for 18 days at 37°C under 5% (vol./vol.) CO2 in medium (DMEM/F12) supplemented with 10% (vol./vol.) FBS (Hiclone; Thermo Fisher Scientific, UT, USA). and 1% (vol./vol.) penicillin/streptomycin (Hiclone; Thermo Fisher Scientific). ARPE-19 cells from passage 20 were used, and the medium was changed every 3-4 days. For permeability studies, ARPE-19 cells were seeded at 400,000 cells/ml (80,000 RPE cells/well) in 0.33 cm<2>HTS-Transwells (Costar; Corning, NY, USA). For real-time PCR, western blot analysis, and immunofluorescence cells were seeded at 20,000 cells/ml.

Experimental conditions and treatments: besides the euglycemic state, eighteen different conditions were tested in cells grown under 25 mmol/l D-glucose:

Condition 1) Control cells did not receive any 5 mm D-glucose treatment

Condition 2) Cells are treated with diabetic milieu 25 mm D-glucose vehicle (Days 15, 16 and 17 per application/day) to evaluate their effect in preventing diabetic milieu-induced cell damage.

Condition 3, 4, 5, 6, 7, 8) Cells were treated with 25 mm D-glucose and two different concentrations of each product: Ic-007a (3, 4) 25 µg/ml (=53 µm) and 50 µg/mL (=107 µm); IIc-007a (5, 6) 25 µg/ml (=53 µm) and 50 µg/ml (=107 µm); IVc-059a (7, 8) 20 µg/ml (=50 µm) and 40 µg/ml (=100 µm) for 72 h (days 15, 16 and 17 per application/day) to evaluate their potential cytotoxic effects. Condition 9) Cells were treated with 25 mm D-glucose and bevacizumab (0.2 mg/ml) for 72 h (days 15, 16 and 17 per application/day) to evaluate their potential cytotoxic effects.

Condition 10) = diabetic milieu; cells were treated with 25 mm D-glucose IL-1β (10 ng/ml) TNF-α (25 ng/ml) VEGF (25 ng/ml) for 48 h (day 16 and 17 in one application/day) to induce monolayer disruption.

Condition 11, 12, 13, 14, 15, 16) Cells were treated with diabetic medium (25 mm D-glucose IL-1β (10 ng/ml) TNF-α (25 ng/ml) VEGF (25 ng) /ml)) two concentrations of novel compounds (Ic-007a, IIc-007a and IVc-059a (15, 16 and 17). day per one application/day) in order to evaluate their effect in the prevention of cell damage caused by the diabetic milieu.

Condition 17) Cells were treated with diabetic medium (25 mm D-glucose IL-1β (10 ng/ml) TNF-α (25 ng/ml) VEGF (25 ng/ml)) bevacizumab

(0.2 mg/ml) (15th, 16th and 17th day per application/day) in order to evaluate their effect in preventing cell damage caused by the diabetic milieu.

ARPE-19 permeability measurement: RPE cell permeability was determined at 18 days by measuring the apical to basolateral movement of FITC-dextran (40 kDa) (Sigma, St Louis, MI, USA) according to the procedure previously reported by Villarroel et al. Exp Eye Res, 2009, 89, 913-920.

Inner blood retinal barrier conditions: primary human retinal endothelial cells (HRECs) were obtained from a vial of cryopreserved cells purchased from Innoprot (Vizcay, Spain). These cells were isolated from human retinal tissue of cadaveric eyes digested with 0.1 mg/ml collagenase type I at 37 °C for 1 hour. Then, endothelial cells were finally selected with magnetic beads coated with CD31 antibody (DynaBeads; Dynal, Oslo, Norway). HRECs were thawed in the laboratory and cultured in endothelial basal medium (EBM) containing 5.5 mm D-glucose, 5% FBS (fetal bovine serum), 100 U/ml penicillin, 100 µg/mL streptomycin and ECG (Endotheli Cell Growth). Supplement) supplement (Innoprot, Vizcaya, Spain). Human fibronectin (MerckMillipore, Madrid, Spain) at 5 µg/ml was used for cell attachment. The cells on paragraphs 2-3 will be used for the experiments. For experimentation, HRECs were grown to confluence, and then the cell culture medium was changed to medium supplemented with 1% FBS for 24 h, and then exposed to different treatments.

Conditions and treatments: The same conditions specified for the experiment in ARPE-19 cells were used. Measurement of HREC permeability: Permeability in HREC monolayers was obtained on permeable supports at 12 × 10<5> cells/well (24 wells, PCF filters, Merck Millipore). The inserts were incubated for 48 hours at 37°C in 5% CO2-air to form a monolayer. Finally, the medium was depleted of serum for 24 h before continuing with the treatments. The lower chamber was filled with 600 µl of complete EBM medium, and the upper chamber with 100 µl of serum-depleted cell suspension (1%).

To detect changes in permeability, 100 µg/ml fluorescent FITC-DEXTRAN (Sigma, Madrid, Spain) was added to the upper side of the insert. Aliquots of 200 µl from the basal compartment were read in a SpectraMak Gemini (Molecular Devices, Sunnivale, CA) at an excitation/emission wavelength of 485/528 nm every 30 min. Finally, the dextran concentration was determined by extrapolation of the fluorescence readings in the standard curve. Each condition was tested in triplicate.

Cell viability and proliferation: Cell counting and MTT assay were performed in HRECs to assess cell viability under the tested conditions. Viability and proliferation of HRECs were measured. Briefly, cells were stained with DAPI and photographs were taken (20x) in a fluorescence inverted microscope (Olympus iX71 with V-RFL-T Olympus). Cell nuclei were counted using Image J software in three different fields for each condition. The experiments were repeated three times. Additionally, the MTT assay (Sigma, Madrid, Spain) was used to assess cell viability.

Briefly, 10 µl of 5 mg/ml MTT in PBS was added to each well after treatment and incubated for an additional 1 h at 37 °C. The medium was removed and the resulting formazan granules were dissolved in 100% dimethyl sulfoxide (DMSO) and absorbance was detected at 562 nm using an ELISA plate reader (ELx800. Bio-Tek Instruments, VT, USA). This assay excluded potential bias in the results due to changes in cell proliferation among different conditions.

Mechanisms of action:

− Proinflammatory cytokines were assessed in ARPE-19 cells by RT-PCR and mRNA for IL-6, TNF-α, IL-1β, IL-18, VEGF, IL-10, FGF were measured using the double delta CT method 2-ΔΔCT with Cts values for the gene of interest and endogenous human B-actin to obtain ΔCT; mRNA values are expressed as relative change compared to the 25 mm D-Gluc condition. (ref: Livak KJ, Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method, Methods, 2001, 25(4), 402-408, doi 10.1006/meth.2001.1262)

− ROS production was assessed using the Cell Biolabs OkiSelect<™>intracellular ROS assay (Green fluorescence) (Bionova, Madrid, Spain). The assay uses the cell-permeable fluorogenic probe 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA), oxidized to highly fluorescent 2', 7'-dichlorodihydrofluorescein (DCF) by cellular ROS.

- The expression of endothelin-1, PDGF and VCAM-1 was analyzed in HREC monolayers by immunohistochemical analysis and RT-PCR.

- TJ (ZO) expression was analyzed in ARPE-19 and HREC monolayers by immunohistochemical analysis.

Results:

The results of the permeability of the outer blood retinal barrier (ARPE19 monolayer) and the inner blood retinal barrier (HREC) are shown in Table 58. Compounds Ic-007a, IIc-007a, IVc-059a reduced the permeability of the outer and inner monolayers.

The effect of compounds Ic-007a, IIc-007a, IVc-059a on the production of ROS and TNF-α, IL-1β, IL-6, IL-18 and VEGF-mRNA in ARPE19 monolayer cells exposed to non-diabetic and diabetic milieu are shown in Table 59.

Table 58: Outer blood retinal barrier and inner blood retinal barrier permeability using FITC-dextran fluorescence leakage (ng/mL/cm<2>).

Table 59: Production of ROS and TNF-a, IL-1b, IL-6, IL-18 and VEGF mRNA by ARPE19 monolayer cells in non-diabetic and diabetic environments.

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Claims (21)

PATENT CLAIMS 1. Substituted hydroquinone of formula (I): whereby: Ra, Rb = H, C1-4acyl, arylC1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl; R1 = COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, or CONH-R10; one of R2 or R3 is H and the other is R5; R4= H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazine-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkyloxycarbonyloxymethyl, C1-C6alkylcarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R5 = R6, R7, or R8 R6 = CONH-R9, CONHCOR9, CONH(CH2)n-R9, CONHCH(COOR4)(CH2)kR9,; where k = 0-4; R7 = benzoheteroaryl substituted with at least one or more groups selected from: COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, OR4, 5-membered heterocycles containing N or fluorine; R8 = (CH2)mX(CH2)pR9; X = O, S, SO2, NH, NAc, or N(CH2)qR9; R9 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered heterocycles containing N, fluorine, C=O, or NHCO-R10; R10 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered N-containing heterocycles, or fluorine; wherein aryl R9 and R10 is an aromatic group containing from 6 to 14 carbon atoms, selected from phenyl, naphthyl, biphenyl groups; and heteroaryl R9 and R10 is an aromatic group containing 1 to 14 carbon atoms and one or more nitrogens; and n, m, p and q are independently 1-4. 2. Compound of formula (I), according to claim 1: whereby: Ra, Rb = H, C1-4acyl, arylC1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl; R1 = COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, or CONH-R10; one of R2 or R3 is H and the other is R5; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R5 = R6, or R7, R6 = CONH-R9, CONHCOR9, CONH(CH2)n-R9, or CONHCH(COOR4)(CH2)kR9; where k = 0-4; R7 = benzoheteroaryl substituted with at least one or more groups selected from: COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, OR4, 5-membered heterocycles containing N or fluorine; R9 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered heterocycles containing N, fluorine, C=O, or NHCO-R10; R10 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered N-containing heterocycles, or fluorine; wherein aryl R9 and R10 is an aromatic group containing from 6 to 14 carbon atoms, selected from phenyl, naphthyl, biphenyl groups; and heteroaryl R9 and R10 is an aromatic group containing 1 to 14 carbon atoms and one or more nitrogens; and n is independently 1-4. 3. The compound according to claim 2, wherein: (A) Ra, Rb = H, C1-4acyl, arylC1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl; R1 = COOR4, R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R 2 = H; R3 = R5; and R5 = R6 = CONH-R9; or (B) Ra, Rb = H, C1-4acyl, arylC1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl; R1 = COOR4, R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazine-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkyloxycarbonyloxymethyl, C1-C6carbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R3 = H; R2 = R5; and R5 = R6 = CONH-R9; or wherein said compound is selected from the group consisting of: 4-(2-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 2,5-dihydroxy-4-(2-sulfophenylaminocarbonyl)benzoic acid; 4-(3-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(3-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 2,5-dihydroxy-4-(3-sulfophenylaminocarbonyl)benzoic acid; 4-(4-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(4-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 2,5-dihydroxy-4-(4-sulfophenylaminocarbonyl)benzoic acid; 4-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(4-carboxy-3-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(4-carboxy-2,5-dihydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(2-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3,5-bis(2,5-dihydroxy-4-carboxybenzoylamino)benzoic acid; 3-(4-carboxy-2,5-dihydroxybenzamido)phthalic acid; 2-(4-carboxy-2,5-dihydroxybenzamido)terephthalic acid; 2-(4-carboxy-2,5-dihydroxybenzamido)isophthalic acid; 4-(4-carboxy-2,5-dihydroxybenzamido)phthalic acid; 5-(4-carboxy-2,5-dihydroxybenzamido)isophthalic acid; 2-(4-carboxy-2,5-dihydroxybenzamido)nicotinic acid; 2-(4-carboxy-2,5-dihydroxybenzamido)isonicotinic acid; 6-(4-carboxy-2,5-dihydroxybenzamido)nicotinic acid; 6-(4-carboxy-2,5-dihydroxybenzamido)picolinic acid; 2-(4-carboxy-2,5-dihydroxybenzamido)-5-fluoronicotinic acid; 6-(4-carboxy-2,5-dihydroxybenzamido)pyridine-2,5-dicarboxylic acid; 2-(4-carboxy-2,5-dihydroxybenzamido)pyridine-3,5-dicarboxylic acid; 3-(4-carboxy-2,5-dihydroxybenzamido)isonicotinic acid; 5-(4-carboxy-2,5-dihydroxybenzamido)nicotinic acid; 5-(4-carboxy-2,5-dihydroxybenzamido)picolinic acid; 3-(4-carboxy-2,5-dihydroxybenzamido)picolinic acid; 5-(4-carboxy-2,5-dihydroxybenzamido)-2-fluoroisonicotinic acid; 4-(4-carboxy-2,5-dihydroxybenzamido)nicotinic acid; 4-(4-carboxy-2,5-dihydroxybenzamido)picolinic acid; 4-(4-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(3-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 2,5-dihydroxy-4-(3-(1-hydroxy-1H-pyrazol-4-yl)phenylaminocarbonyl)benzoic acid; 4-(2-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; ethyl 4-(2-(ethoxycarbonyl)phenylaminocarbonyl)-2,5-diacetoxybenzoate; ethyl 4-(2-(ethoxycarbonyl)phenylaminocarbonyl)-2,5-dihydroxybenzoate; ethyl 4-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-diacetoxybenzoate; ethyl 4-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoate; ethyl 2,5-dihydroxy-4-(4-hydroxy-3-(methoxycarbonyl)phenylaminocarbonyl)benzoate; ethyl 3,5-bis(2,5-diacetoxy-4-ethoxycarbonylbenzoylamino)benzoate; ethyl 3,5-bis(2,5-dihydroxy-4-ethoxycarbonylbenzoylamino)benzoate; ethyl 3-(4-(ethoxycarbonyl)-2,5-dihydroxybenzamido)isonicotinate; ethyl 4-(4-ethoxycarbonyl-2,5-dihydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoate; 3-(2-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; ,5-dihydroxy-3-(2-sulfophenylaminocarbonyl)benzoic acid; -(3-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; -(3-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; ,5-dihydroxy-3-(3-sulfophenylaminocarbonyl)benzoic acid; -(4-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; -(4-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; ,5-dihydroxy-3-(4-sulfophenylaminocarbonyl)benzoic acid; -(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; -(4-carboxy-3-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; -(2-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzoic acid; ,5-bis(2,5-dihydroxy-3-carboxybenzoylamino)benzoic acid; -(3-carboxy-2,5-dihydroxybenzamido)phthalic acid; -(3-carboxy-2,5-dihydroxybenzamido)terephthalic acid; -(3-carboxy-2,5-dihydroxybenzamido)isophthalic acid; -(3-carboxy-2,5-dihydroxybenzamido)phthalic acid; -(3-carboxy-2,5-dihydroxybenzamido)isophthalic acid; -(3-carboxy-2,5-dihydroxybenzamido)nicotinic acid; -(3-carboxy-2,5-dihydroxybenzamido)isonicotinic acid; -(3-carboxy-2,5-dihydroxybenzamido)nicotinic acid; -(3-carboxy-2,5-dihydroxybenzamido)picolinic acid; -(3-carboxy-2,5-dihydroxybenzamido)-5-fluoronicotinic acid; 6-(3-carboxy-2,5-dihydroxybenzamido)pyridine-2,5-dicarboxylic acid; 2-(3-carboxy-2,5-dihydroxybenzamido)pyridine-3,5-dicarboxylic acid; 3-(3-carboxy-2,5-dihydroxybenzamido)isonicotinic acid; 5-(3-carboxy-2,5-dihydroxybenzamido)nicotinic acid; 5-(3-carboxy-2,5-dihydroxybenzamido)picolinic acid; 3-(3-carboxy-2,5-dihydroxybenzamido)picolinic acid; 5-(3-carboxy-2,5-dihydroxybenzamido)-2-fluoroisonicotinic acid; 4-(3-carboxy-2,5-dihydroxybenzamido)nicotinic acid; 4-(3-carboxy-2,5-dihydroxybenzamido)picolinic acid; 3-(4-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3-(3-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; 2,5-dihydroxy-3-(3-(1-hydroxy-1H-pyrazol-4-yl)phenylaminocarbonyl)benzoic acid; 3-(2-(carboxymethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoic acid; ethyl 3-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzoate; and ethyl 3-(2-(ethoxycarbonylmethyl)phenylaminocarbonyl)-2,5-dihydroxybenzoate; 4. The compound according to claim 2, wherein: (A) Ra, Rb = H; R1 = SO3H; R 2 = H; R3 = R5; and R5 = R6 = CONH-R9; or (B) Ra, Rb = H; R1 = SO3H; R3 = H; R2 = R5; and R5 = R6 = CONH-R9; or wherein said compound is selected from the group consisting of: 2-(2,5-dihydroxy-4-sulfobenzamido)benzoic acid; 2,5-dihydroxy-4-(2-sulfophenylaminocarbonyl)benzenesulfonic acid; 3-(2,5-dihydroxy-4-sulfobenzamido)benzoic acid; 2,5-dihydroxy-4-(3-sulfophenylaminocarbonyl)benzenesulfonic acid; 4-(2,5-dihydroxy-4-sulfobenzamido)benzoic acid; 2,5-dihydroxy-4-(4-sulfophenylaminocarbonyl)benzenesulfonic acid; 5-(2,5-dihydroxy-4-sulfobenzamido)-2-hydroxybenzoic acid; 4-(2,5-dihydroxy-4-sulfobenzamido)-2-hydroxybenzoic acid; 2-(2,5-dihydroxy-4-sulfobenzamido)-5-hydroxybenzoic acid; 3,5-bis(2,5-dihydroxy-4-sulfobenzamido)benzoic acid; 3-(2,5-dihydroxy-4-sulfobenzamido)phthalic acid; 2-(2,5-dihydroxy-4-sulfobenzamido)terephthalic acid; 2-(2,5-dihydroxy-4-sulfobenzamido)isophthalic acid; 4-(2,5-dihydroxy-4-sulfobenzamido)phthalic acid; 5-(2,5-dihydroxy-4-sulfobenzamido)isophthalic acid; 2-(2,5-dihydroxy-4-sulfobenzamido)nicotinic acid; 2-(2,5-dihydroxy-4-sulfobenzamido)isonicotinic acid; 6-(2,5-dihydroxy-4-sulfobenzamido)nicotinic acid; 6-(2,5-dihydroxy-4-sulfobenzamido)picolinic acid; 2-(2,5-dihydroxy-4-sulfobenzamido)-5-fluoronicotinic acid; 6-(2,5-dihydroxy-4-sulfobenzamido)pyridine-2,5-dicarboxylic acids; 2-(2,5-dihydroxy-4-sulfobenzamido)pyridine-3,5-dicarboxylic acids; 3-(2,5-dihydroxy-4-sulfobenzamido)isonicotinic acid; 5-(2,5-dihydroxy-4-sulfobenzamido)nicotinic acid; 5-(2,5-dihydroxy-4-sulfobenzamido)picolinic acid; 3-(2,5-dihydroxy-4-sulfobenzamido)picolinic acid; 5-(2,5-dihydroxy-4-sulfobenzamido)-2-fluoroisonicotinic acid; 4-(2,5-dihydroxy-4-sulfobenzamido)nicotinic acid; 4-(2,5-dihydroxy-4-sulfobenzamido)picolinic acid; (4-(2,5-dihydroxy-4-sulfobenzamido)phenyl)acetic acid; (3-(2,5-dihydroxy-4-sulfobenzamido)phenyl)acetic acid; (2-(2,5-dihydroxy-4-sulfobenzamido)phenyl)acetic acid; 2-(2,5-dihydroxy-3-sulfobenzamido)benzoic acid; 2,5-dihydroxy-3-(2-sulfophenylaminocarbonyl)benzenesulfonic acid; 3-(2,5-dihydroxy-3-sulfobenzamido)benzoic acid; 2,5-dihydroxy-3-(3-sulfophenylaminocarbonyl)benzenesulfonic acid; 4-(2,5-dihydroxy-3-sulfobenzamido)benzoic acid; 2,5-dihydroxy-3-(4-sulfophenylaminocarbonyl)benzenesulfonic acid; 5-(2,5-dihydroxy-3-sulfobenzamido)-2-hydroxybenzoic acid; 4-(2,5-dihydroxy-3-sulfobenzamido)-2-hydroxybenzoic acid; 2-(2,5-dihydroxy-3-sulfobenzamido)-5-hydroxybenzoic acid; 3-(2,5-dihydroxy-3-sulfobenzamido)-5-(2,5-dihydroxy-4-sulfobenzamido)benzoic acid 3-(2,5-dihydroxy-3-sulfobenzamido)phthalic acid; 2-(2,5-dihydroxy-3-sulfobenzamido)terephthalic acid; 2-(2,5-dihydroxy-3-sulfobenzamido)isophthalic acid; 4-(2,5-dihydroxy-3-sulfobenzamido)phthalic acid; 5-(2,5-dihydroxy-3-sulfobenzamido)isophthalic acid; 2-(2,5-dihydroxy-3-sulfobenzamido)nicotinic acid; 2-(2,5-dihydroxy-3-sulfobenzamido)isonicotinic acid; 6-(2,5-dihydroxy-3-sulfobenzamido)nicotinic acid; 6-(2,5-dihydroxy-3-sulfobenzamido)picolinic acid; 2-(2,5-dihydroxy-3-sulfobenzamido)-5-fluoronicotinic acid; 6-(2,5-dihydroxy-3-sulfobenzamido)pyridine-2,5-dicarboxylic acids; 2-(2,5-dihydroxy-3-sulfobenzamido)pyridine-3,5-dicarboxylic acids; 3-(2,5-dihydroxy-3-sulfobenzamido)isonicotinic acid; 5-(2,5-dihydroxy-3-sulfobenzamido)nicotinic acid; 5-(2,5-dihydroxy-3-sulfobenzamido)picolinic acid; 3-(2,5-dihydroxy-3-sulfobenzamido)picolinic acid; 5-(2,5-dihydroxy-3-sulfobenzamido)-2-fluoroisonicotinic acid; 4-(2,5-dihydroxy-3-sulfobenzamido)nicotinic acid; 4-(2,5-dihydroxy-3-sulfobenzamido)picolinic acid; (4-(2,5-dihydroxy-3-sulfobenzamido)phenyl)acetic acid; (3-(2,5-dihydroxy-3-sulfobenzamido)phenyl)acetic acid; and (2-(2,5-dihydroxy-3-sulfobenzamido)phenyl)acetic acid. 5. The compound according to claim 2, wherein: (A) Ra, Rb = H, R1 = (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R 2 = H or R 5 , R 3 = R 5 ; and R5 = R6 = CONH-R9; or (B) Ra, Rb = H; R1 = (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazine-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy1-ethyl, C1-C6alkyloxycarbonyloxymethyl, C1-C6alkyloxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R3 = H; R2 = R5; and R5 = R6 = CONH-R9; or wherein said compound is selected from the group consisting of: 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)benzoic acid; (2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (2,5-dihydroxy-4-(2-sulfophenylaminocarbonyl)phenyl)acetic acid; 3-(4-(carboxymethyl)-2,5-dihydroxybenzamido)benzoic acid; (2,5-dihydroxy-4-(3-sulfophenylaminocarbonyl)phenyl)acetic acid; 4-(4-(carboxymethyl)-2,5-dihydroxybenzamido)benzoic acid; 2-(2,5-dihydroxy-4-(4-sulfophenylaminocarbonyl)phenyl)acetic acid; 5-(4-(carboxymethyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid; 4-(4-(carboxymethyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid; 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)-5-hydroxybenzoic acid; 3,5-bis(2,5-dihydroxy-4-carboxymethylbenzoylamino)benzoic acid 3-(4-(carboxymethyl)-2,5-dihydroxybenzamido)phthalic acid; 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)terephthalic acid; 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)isophthalic acid; 4-(4-(carboxymethyl)-2,5-dihydroxybenzamido)phthalic acid; 5-(4-(carboxymethyl)-2,5-dihydroxybenzamido)isophthalic acid; 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)nicotinic acid; 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)isonicotinic acid; 6-(4-(carboxymethyl)-2,5-dihydroxybenzamido)nicotinic acid; 6-(4-(carboxymethyl)-2,5-dihydroxybenzamido)picolinic acid; 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)-5-fluoronicotinic acid; 6-(4-(carboxymethyl)-2,5-dihydroxybenzamido)pyridine-2,5-dicarboxylic acid; 2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)pyridine-3,5-dicarboxylic acids; 3-(4-(carboxymethyl)-2,5-dihydroxybenzamido)isonicotinic acid; 5-(4-(carboxymethyl)-2,5-dihydroxybenzamido)nicotinic acid; 5-(4-(carboxymethyl)-2,5-dihydroxybenzamido)picolinic acid; 3-(4-(carboxymethyl)-2,5-dihydroxybenzamido)picolinic acid; 5-(4-(carboxymethyl)-2,5-dihydroxybenzamido)-2-fluoroisonicotinic acid; 4-(4-(carboxymethyl)-2,5-dihydroxybenzamido)nicotinic acid; 4-(4-(carboxymethyl)-2,5-dihydroxybenzamido)picolinic acid; (4-(4-(carboxymethyl)-2,5-dihydroxybenzamido)phenyl)acetic acid; (3-(4-(carboxymethyl)-2,5-dihydroxybenzamido)phenyl)acetic acid; (2-(4-(carboxymethyl)-2,5-dihydroxybenzamido)phenyl)acetic acid; methyl 2-(4-(ethoxycarbonylmethyl)-2,5-dihydroxybenzamido)benzoate; ethyl (4-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxyphenyl)acetate; diethyl 5-(4-(ethoxycarbonylmethyl)-2,5-dihydroxybenzamido)isophthalate; methyl 3,5-bis(4-(ethoxycarbonylmethyl)-2,5-dihydroxybenzamido)benzoate; 2-(3-(carboxymethyl)-2,5-dihydroxybenzamido)benzoic acid; (2,5-dihydroxy-3-(2-sulfophenylaminocarbonyl)phenyl)acetic acid; 3-(3-(carboxymethyl)-2,5-dihydroxybenzamido)benzoic acid; (2,5-dihydroxy-3-(3-sulfophenylaminocarbonyl)phenyl)acetic acid; 4-(3-(carboxymethyl)-2,5-dihydroxybenzamido)benzoic acid; (2,5-dihydroxy-3-(4-sulfophenylaminocarbonyl)phenyl)acetic acid; 5-(3-(carboxymethyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid; 4-(3-(carboxymethyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid; 2-(3-(carboxymethyl)-2,5-dihydroxybenzamido)-5-hydroxybenzoic acid; 3,5-bis(2,5-dihydroxy-3-carboxymethylbenzoylamino)benzoic acid 3-(3-(carboxymethyl)-2,5-dihydroxybenzamido)phthalic acid; 2-(3-(carboxymethyl)-2,5-dihydroxybenzamido)terephthalic acid; 2-(3-(carboxymethyl)-2,5-dihydroxybenzamido)isophthalic acid; 4-(3-(carboxymethyl)-2,5-dihydroxybenzamido)phthalic acid; 5-(3-(carboxymethyl)-2,5-dihydroxybenzamido)isophthalic acid; 2-(3-(carboxymethyl)-2,5-dihydroxybenzamido)nicotinic acid; 2-(3-(carboxymethyl)-2,5-dihydroxybenzamido)isonicotinic acid; 6-(3-(carboxymethyl)-2,5-dihydroxybenzamido)nicotinic acid; 6-(3-(carboxymethyl)-2,5-dihydroxybenzamido)picolinic acid; 2-(3-(carboxymethyl)-2,5-dihydroxybenzamido)-5-fluoronicotinic acid; 6-(3-(carboxymethyl)-2,5-dihydroxybenzamido)pyridine-2,5-dicarboxylic acid; 2-(3-(carboxymethyl)-2,5-dihydroxybenzamido)pyridine-3,5-dicarboxylic acids; 3-(3-(carboxymethyl)-2,5-dihydroxybenzamido)isonicotinic acid; 5-(3-(carboxymethyl)-2,5-dihydroxybenzamido)nicotinic acid; 5-(3-(carboxymethyl)-2,5-dihydroxybenzamido)picolinic acid; 3-(3-(carboxymethyl)-2,5-dihydroxybenzamido)picolinic acid; 5-(3-(carboxymethyl)-2,5-dihydroxybenzamido)-2-fluoroisonicotinic acid; 4-(3-(carboxymethyl)-2,5-dihydroxybenzamido)nicotinic acid; 4-(3-(carboxymethyl)-2,5-dihydroxybenzamido)picolinic acid; (4-(3-(carboxymethyl)-2,5-dihydroxybenzamido)phenyl)acetic acid; (3-(3-(carboxymethyl)-2,5-dihydroxybenzamido)phenyl)acetic acid; and (2-(3-(carboxymethyl)-2,5-dihydroxybenzamido)phenyl)acetic acid. 6. The compound according to claim 2, wherein: (A) Ra, Rb = H, C1-4acyl, arylC1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl; R1 = CONH-R10; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-ceacyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R 2 = H; R3 = R5; and R5 = R6 = CONH-R9; or (B) Ra, Rb = H, C1-4acyl, arylC1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl; R1 = CONH-R10; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-cealkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R3 = H; R2 = R5; and R5 = R6 = CONH-R9. or wherein said compound is selected from the group consisting of: 5-(4-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid; N1,N4-bis(2-(1H-tetrazol-5-yl)phenyl)-2,5-dihydroxyterephthalamide; 5-(4-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid; 5-(2,5-dihydroxy-4-(4-hydroxy-3-sulfophenylaminocarbonyl)benzamido)-2-hydroxybenzenesulfonic acid; 4-(4-(4-carboxy-3-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid; 4-(2,5-dihydroxy-4-(3-hydroxy-4-sulfophenylaminocarbonyl)benzamido)-2-hydroxybenzenesulfonic acid; 2-(2,5-dihydroxy-4-(4-hydroxy-2-carboxyphenylaminocarbonyl)benzamido)-5-hydroxybenzoic acid; 2-(2,5-dihydroxy-4-(4-hydroxy-2-sulfophenylaminocarbonyl)benzamido)-5-hydroxybenzene sulfonic acid; methyl 5-(4-(2-(1H-tetrazol-5-yl)phenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoate; methyl 5-(2,5-diacetoxy-4-(4-acetoxy-3-(methoxycarbonyl)phenylaminocarbonyl)benzamido)-2-acetoxybenzoate; methyl 5-(2,5-dihydroxy-4-(4-hydroxy-3-(methoxycarbonyl)phenylaminocarbonyl)benzamido)-2-hydroxybenzoate; methyl 2-(2,5-dihydroxy-4-(4-hydroxy-2-(methoxycarbonyl)phenylaminocarbonyl)benzamido)-5-hydroxybenzoate; 1-(2,2-dimethylpropanoyloxy)ethyl 5-[[4-[[3-[1-(2,2-dimethylpropanoyloxy)ethoxycarbonyl]-4-hydroxy-phenyl]aminocarbonyl]-2,5-dihydroxybenzoyl]amino]-2-hydroxybenzoate 5-(3-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid; 5-(2,5-dihydroxy-3-(4-hydroxy-3-sulfophenylaminocarbonyl)benzamido)-2-hydroxybenzenesulfonic acid; 4-(3-(3-hydroxy-4-carboxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid; 4-(2,5-dihydroxy-3-(3-hydroxy-4-sulfophenylaminocarbonyl)benzamido)-2-hydroxybenzenesulfonic acid; 2-(3-(2-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-5-hydroxylbenzoic acid; and 2-(2,5-dihydroxy-3-(4-hydroxy-2-sulfophenylaminocarbonyl)benzamido)-5-hydroxybenzenesulfonic acid. 7. A compound according to claim 2 having the formula (I): whereby: Ra, Rb = H; R1 = CONH-R10; R10 = phenyl substituted with COOR4 and with OR4; R 2 = H; R3 = CONH-R9; R9 = phenyl substituted with COOR4 and with OR4; and R4 = H; and wherein the compound is 5-(4-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid. 8. A compound according to claim 2 having the formula (I): whereby: Ra, Rb = H; R1 = CONH-R10; R10 = phenyl substituted with COOR4 and with OR4; R2 = CONH-R9; R3 = H; R9 = phenyl substituted with COOR4 and with OR4; and R4 = H; and wherein the compound is 5-(3-(3-carboxy-4-hydroxyphenylaminocarbonyl)-2,5-dihydroxybenzamido)-2-hydroxybenzoic acid. 9. The compound according to claim 2, wherein: (A) Ra, Rb = H; R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R 2 = H; R3 = R5; and R5 = R6 = CONHCOR9; or (B) Ra, Rb = H; R<1>= COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R3 = H; R2 = R5; and R5 = R6 = CONHCOR9; or wherein said compound is selected from the group consisting of: N-(1,3,4,5-tetrahydroxycyclohexylcarbonyl) 4-carboxy-2,5-dihydroxybenzamide; N-(4-carboxy-2,5-dihydroxybenzoyl) 4-carboxy-2,5-dihydroxy-benzamide; N-(3-carboxy-2,5-dihydroxybenzoyl) 4-carboxy-2,5-dihydroxy-benzamide; N-(3,4-dihydroxybenzoyl) 4-carboxy-2,5-dihydroxybenzamide; N-(3,5-dihydroxybenzoyl) 4-carboxy-2,5-dihydroxybenzamide; N-(1,3,4,5-tetrahydroxycyclohexylcarbonyl) 3-carboxy-2,5-dihydroxybenzamide; N-(4-carboxy-2,5-dihydroxybenzoyl) 3-carboxy-2,5-dihydroxy-benzamide; N-(3-carboxy-2,5-dihydroxybenzoyl) 3-carboxy-2,5-dihydroxy-benzamide; N-(3,4-dihydroxybenzoyl) 3-carboxy-2,5-dihydroxybenzamide; N-(3,5-dihydroxybenzoyl) 3-carboxy-2,5-dihydroxybenzamide; N-(1,3,4,5-tetrahydroxycyclohexylcarbonyl) 4-carboxymethyl-2,5-dihydroxybenzamide; N-(4-carboxy-2,5-dihydroxybenzoyl) 4-carboxymethyl-2,5-dihydroxybenzamide; N-(3-carboxy-2,5-dihydroxybenzoyl) 4-carboxymethyl-2,5-dihydroxybenzamide; N-(3,4-dihydroxybenzoyl) 4-carboxymethyl-2,5-dihydroxybenzamide; N-(3,5-dihydroxybenzoyl) 4-carboxymethyl-2,5-dihydroxybenzamide; N-(1,3,4,5-tetrahydroxycyclohexylcarbonyl) 3-carboxymethyl-2,5-dihydroxybenzamide; N-(4-carboxy-2,5-dihydroxybenzoyl) 3-carboxymethyl-2,5-dihydroxybenzamide; N-(3-carboxy-2,5-dihydroxybenzoyl) 3-carboxymethyl-2,5-dihydroxybenzamide; N-(3,4-dihydroxybenzoyl) 3-carboxymethyl-2,5-dihydroxybenzamide; and N-(3,5-dihydroxybenzoyl) 3-carboxymethyl-2,5-dihydroxybenzamide. 10. The compound according to claim 2, wherein: (A) Ra, Rb = H; R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R 2 = H; R3 = R5; and R5 = R6 = CONH(CH2)n-R9; or (B) Ra, Rb = H; R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R3 = H; R2 = R5; and R5 = R6 = CONH(CH2)n-R9; or wherein said compound is selected from the group consisting of: 4-(3,4-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(2-(3,4-dihydroxyphenyl)ethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(3,5-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-((4,5-dihydroxy-3-oxocyclohex-1-enyl)methylaminocarbonyl)-2,5-dihydroxybenzoic acid; 2,5-dihydroxy-4-((3,4,5-trihydroxycyclohexyl)methylaminocarbonyl)benzoic acid; 4-(2-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(3-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(4-carboxyphenylmethylaminocarbonyl) 2,5-dihydroxybenzoic acid; 3-(3,4-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3-(2-(3,4-dihydroxyphenyl)ethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3-(3,5-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3-((4,5-dihydroxy-3-oxocyclohex-1-enyl)methylaminocarbonyl)-2,5-dihydroxybenzoic acid; 2,5-dihydroxy-3-((3,4,5-trihydroxycyclohexyl)methylaminocarbonyl)benzoic acid; 3-(2-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3-(3-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3-(4-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; (4-(3,4-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (4-(3,5-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (4-((4,5-dihydroxy-3-oxocyclohex-1-enyl)methylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (2,5-dihydroxy-4-((3,4,5-trihydroxycyclohexyl)methylaminocarbonyl)phenyl)acetic acid; (4-(2-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (4-(3-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (4-(4-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (3-(3,4-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (3-(3,5-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (3-((4,5-dihydroxy-3-oxocyclohex-1-enyl)methylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (2,5-dihydroxy-3-((3,4,5-trihydroxycyclohexyl)methylcarbonylamino)phenyl)acetic acid; (3-(2-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (3-(3-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; and (3-(4-carboxyphenylmethylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid. 11. The compound according to claim 2, characterized in that the compound is selected from the group consisting of: N-(1,3,4,5-tetrahydroxycyclohexylcarbonyl) 4-carboxy-2,5-dihydroxybenzamide; N-(1,3,4,5-tetrahydroxycyclohexylcarbonyl) 3-carboxy-2,5-dihydroxybenzamide; N-(1,3,4,5-tetrahydroxycyclohexylcarbonyl) 4-carboxymethyl-2,5-dihydroxybenzamide; N-(1,3,4,5-tetrahydroxycyclohexylcarbonyl) 3-carboxymethyl-2,5-dihydroxybenzamide; 4-((4,5-dihydroxy-3-oxocyclohex-1-enyl)methylaminocarbonyl)-2,5-dihydroxybenzoic acid; 2,5-dihydroxy-4-((3,4,5-trihydroxycyclohexyl)methylaminocarbonyl)benzoic acid; 3-(3,5-dihydroxyphenylmethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3-((4,5-dihydroxy-3-oxocyclohex-1-enyl)methylaminocarbonyl)-2,5-dihydroxybenzoic acid; (4-((4,5-dihydroxy-3-oxocyclohex-1-enyl)methylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; (2,5-dihydroxy-4-((3,4,5-trihydroxycyclohexyl)methylaminocarbonyl)phenyl)acetic acid; (3-((4,5-dihydroxy-3-oxocyclohex-1-enyl)methylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; and (2,5-dihydroxy-3-((3,4,5-trihydroxycyclohexyl)methylcarbonylamino)phenyl)acetic acid. 12. The compound according to claim 2, wherein: Ra, Rb = H; R1 = SO3H; R 2 = H; R3 = R5; and R5 = R6 = CONH(CH2)n-R9; or wherein said compound is selected from the group consisting of: 2-((2,5-dihydroxy-4-sulfobenzamido)methyl)benzoic acid; 3-((2,5-dihydroxy-4-sulfobenzamido)methyl)benzoic acid; and 4-((2,5-dihydroxy-4-sulfobenzamido)methyl)benzoic acid. 13. The compound according to claim 2, wherein: (A) Ra, Rb = H; R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-cealkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R 2 = H; R3 = R5; and R5 = R6 = CONHCH(COOR4)(CH2)kR9; or (B) Ra, Rb = H; R1 = COOR4; (CH2)nCOOR4; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R3 = H; R2 = R5; and R5 = R6 = CONHCH(COOR4)(CH2)kR9; or wherein said compound is selected from the group consisting of: 4-(1-carboxy-2-(3,4-dihydroxyphenyl)ethylaminocarbonyl)-2,5-dihydroxybenzoic acid; 4-(carboxy(3,4-dihydroxyphenyl)methylaminocarbonyl)-2,5-dihydroxybenzoic acid; 3-(1-carboxy-2-(3,4-dihydroxyphenyl)ethylaminocarbonyl)-2,5-dihydroxybenzoic acid 3-(carboxy(3,4-dihydroxyphenyl)methylaminocarbonyl)-2,5-dihydroxybenzoic acid; (4-(carboxy(3,4-dihydroxyphenyl)methylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid; and (3-(Carboxy(3,4-dihydroxyphenyl)methylaminocarbonyl)-2,5-dihydroxyphenyl)acetic acid. 14. The compound according to claim 2, wherein: (A) Ra, Rb = H; R1 = COOR4, (CH2)nCOOR4, SO5H, (CH2)nSO3H, CONH-R10; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R 2 = H; R3 = R5; and R5 = R7 = benzoheteroaryl substituted with at least one or more groups selected from: COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, OR4, 5-membered heterocycles containing N or fluorine; or (B) Ra, Rb = H; R1 = COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, CONH-R10; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R3 = H; R2 = R5; and R5 = R7 = benzoheteroaryl substituted with at least one or more groups selected from: COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, OR4, 5-membered heterocycles containing N or fluorine; or wherein said compound is selected from the group consisting of: 2-(4-carboxy-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-4-carboxylic acid; methyl 2-(4-ethoxycarbonyl-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-4-carboxylate; 2-(2,5-dihydroxy-4-sulfophenyl)-1H-benzo[d]imidazole-4-carboxylic acid; 2-(4-carboxy-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid; 2-(2,5-dihydroxy-4-sulfophenyl)-1H-benzo[d]imidazole-5-carboxylic acid; 2-(3-carboxy-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-4-carboxylic acid; 2-(2,5-dihydroxy-3-sulfophenyl)-1H-benzo[d]imidazole-4-carboxylic acid; 2-(3-carboxy-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid; 2-(2,5-dihydroxy-3-sulfophenyl)-1H-benzo[d]imidazole-5-carboxylic acid; 2-(4-(carboxymethyl)-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-4-carboxylic acid; 2-(4-(carboxymethyl)-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid; 2-(3-(carboxymethyl)-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-4-carboxylic acid; and 2-(3-(carboxymethyl)-2,5-dihydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acids. 15. Compound of formula (I), according to claim 1: whereby: Ra, Rb = H, C1-4acyl, arylC1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl; R1 = COOR4, (CH2)nCOOR4, SO5H, (CH2)nSO3H, or CONH-R10; one of R2 or R3 is H and the other is R5; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R5 = R8 R8 = (CH2)mX(CH2)pR9; X = O, S, SO2, NH, NAc, or N(CH2)qR9; R9 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered heterocycles containing N, fluorine, C=O, or NHCO-R10; R10 = aryl, heteroaryl, substituted with at least one or more groups selected from COOR4, (CH2)nCOOR4, (CH2)nSO3H, OR4, SO3H, 5-membered N-containing heterocycles, or fluorine; wherein aryl R9 and R10 is an aromatic group containing from 6 to 14 carbon atoms, selected from a phenyl, naphthyl or biphenyl group; and heteroaryl R9 and R10 is an aromatic group containing 1 to 14 carbon atoms and one or more nitrogens; and n, m, p and k are integers independently equal to 1-4. 16. The compound according to claim 14, wherein: (A) Ra, Rb = H, C1-4acyl, arylC1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl; R1 = COOR4, (CH2)nCOOR4, SO5H, (CH2)nSO3H, CONH-R10; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R 2 = H; R3 = R5; and R5 = R8 = (CH2)mX(CH2)pR9; where X = O, S, SO2, NH, NAc or N(CH2)qR9; or (B) Ra, Rb = H, C1-4acyl, arylC1-4alkyl, C6-C10arylCO, HOOC(CH2)nCO, C1-C7alkylNHCO, phosphono, phosphonooxymethyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl or C1-C6alkoxycarbonyloxy-1-ethyl; R1 = COOR4, (CH2)nCOOR4, SO3H, (CH2)nSO3H, CONH-R10; R4 = H, C1-4alkyl, arylC1-4alkyl, morpholino-C1-C6alkyl, pyrrolidino-C1-C6alkyl, N-methylpiperazino-C1-C6alkyl, C1-C6acyloxymethyl, C1-C6acyloxy-1-ethyl, C1-C6alkoxycarbonyloxymethyl, C1-C6alkoxycarbonyloxy-1-ethyl, or (oxodioxolyl)methyl; o-methoxyphenyl (guaiacol ester); R3 = H; R2 = R5; and R5 = R8 = (CH2)mX(CH2)pR9; where X = O, S, SO2, NH, NAc or N(CH2)qR9; or wherein said compound is selected from the group consisting of: bis(4-carboxy-2,5-dihydroxyphenylmethyl)amine; 4-((2,5-dihydroxy-4-sulfophenylmethylamino)methyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-3-sulfophenylmethylamino)methyl)-2,5-dihydroxybenzoic acid; bis(2,5-dihydroxy-4-sulfophenylmethyl)amine; N,N-bis(4-carboxy-2,5-dihydroxyphenylmethyl)acetamide; N-(2,5-dihydroxy-4-sulfophenylmethyl) N-(4-carboxy-2,5-dihydroxyphenylmethyl)acetamide; N-(2,5-dihydroxy-3-sulfophenylmethyl) N-(4-carboxy-2,5-dihydroxyphenylmethyl)acetamide; N,N-bis(2,5-dihydroxy-4-sulfophenylmethyl)acetamide; 4-((2,5-dihydroxy-4-carboxyphenyl)methylthiomethyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-4-sulfophenyl)methylthiomethyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-3-sulfophenyl)methylthiomethyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-4-sulfophenyl)methylthiomethyl)-2,5-dihydroxybenzenesulfonic acid; 4-((2,5-dihydroxy-4-carboxyphenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-4-sulfophenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-3-sulfophenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-4-sulfophenyl)methylsulfonylmethyl)-2,5-dihydroxybenzenesulfonic acid; 4-((2,5-dihydroxy-4-carboxyphenyl)methoxymethyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-4-sulfophenyl)methoxymethyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-3-sulfophenyl)methoxymethyl)-2,5-dihydroxybenzoic acid; 4-((2,5-dihydroxy-4-sulfophenyl)methoxymethyl)-2,5-dihydroxybenzenesulfonic acid; Tris (4-carboxy-2,5-dihydroxyphenylmethyl)amine; Tris (4-ethoxycarbonyl-2,5-dihydroxyphenylmethyl)amine; Bis(3-carboxy-2,5-dihydroxyphenylmethyl)amine; 3-((2,5-dihydroxy-3-sulfophenylmethylamino)methyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-4-sulfophenylmethylamino)methyl)-2,5-dihydroxybenzoic acid; Bis(2,5-dihydroxy-3-sulfophenylmethyl)amine; N,N-bis(3-carboxy-2,5-dihydroxyphenylmethyl)acetamide; N-(2,5-dihydroxy-3-sulfophenylmethyl) N-(3-carboxy-2,5-dihydroxyphenylmethyl)acetamide; N-(2,5-dihydroxy-4-sulfophenylmethyl) N-(3-carboxy-2,5-dihydroxyphenylmethyl)acetamide; N,N-bis(2,5-dihydroxy-3-sulfophenylmethyl)acetamide; 3-((2,5-dihydroxy-3-carboxyphenyl)methylthiomethyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-3-sulfophenyl)methylthiomethyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-4-sulfophenyl)methylthiomethyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-3-sulfophenyl)methylthiomethyl)-2,5-dihydroxybenzenesulfonic acid; 3-((2,5-dihydroxy-3-carboxyphenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-3-sulfophenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-4-sulfophenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-3-sulfophenyl)methylsulfonylmethyl)-2,5-dihydroxybenzenesulfonic acid; 3-((2,5-dihydroxy-3-carboxyphenyl)methoxymethyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-3-sulfophenyl)methoxymethyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-4-sulfophenyl)methoxymethyl)-2,5-dihydroxybenzoic acid; 3-((2,5-dihydroxy-3-sulfophenyl)methoxymethyl)-2,5-dihydroxybenzenesulfonic acid; methyl 3-((2,5-diacetoxy-3-methoxycarbonylphenyl)methylsulfonylmethyl)-2,5-diacetoxybenzoate; methyl 3-((2,5-dihydroxy-3-methoxycarbonylphenyl)methylsulfonylmethyl)-2,5-hydroxybenzoate; 2-morpholinoethyl 3-((2,5-dihydroxy-3-(2-morpholinoethoxycarbonyl)phenyl)methylsulfonylmethyl)-2,5-hydroxybenzoate; tris (3-carboxy-2,5-dihydroxyphenylmethyl)amine; and tris (3-ethoxycarbonyl-2,5-dihydroxyphenylmethyl)amine. 17. A compound according to claim 2 having the formula (I): whereby: Ra, Rb = H; R1 = COOR4; R 2 = H; R3 = R8; R4 = H; R8 = (CH2)mX(CH2)pR9; R9 = phenyl substituted with COOR4 and with OR4; X = N(CH2)qR9; and m, p and k = 1; and wherein the compound is tris(4-carboxy-2,5-dihydroxyphenylmethyl)amine. 18. A compound according to claim 2 having the formula (I): whereby: Ra, Rb = H; R1 = COOR4; R2 = (CH2)mX(CH2)pR9 R3 = H; R9 = phenyl substituted with COOR4 and with OR4; R4 = H; X = SOz; and m and p = 1; and the compound is 3-((2,5-dihydroxy-3-carboxyphenyl)methylsulfonylmethyl)-2,5-dihydroxybenzoic acid. 19. A pharmaceutical composition containing a therapeutically effective amount of a compound according to any one of claims 1-18 or a pharmaceutically acceptable salt, or stereoisomer thereof, and a pharmaceutically acceptable excipient. 20. A compound according to any one of claims 1-19 for use in the treatment and/or prevention of autoimmune, immunological, rheumatological, vascular disorders, ophthalmological disorders, fibrotic disorders, metabolic and gastrointestinal disorders, neuroinflammatory and neurodegenerative diseases, neoplasms and cancer-related disorders, hormone-related diseases or immune disorders resulting from viral and/or bacterial infectious diseases and/or their complications. 21. Intermediates in the synthesis of compounds according to claim 2, wherein the intermediates are selected from 2,5-bis(benzyloxy)-4-(ethoxycarbonyl)benzoic acid; 2,5-bis(benzyloxy)isophthalaldehyde; 2,5-bis(benzyloxy)isophthalic acid; ethyl 2,5-bis(benzyloxy)-4-(hydroxymethyl)benzoate; or ethyl 2,5-bis(benzyloxy)-3-(hydroxymethyl)benzoate.